Methods for detecting target nucleic acids

Abstract

Provided is a method for detecting target nucleic acid, in particular, a method for using a universal probe to realize single- or multiplexed detection of target nucleic acid in digital PCR. The detection method can realize single-tube super-multiplexed detection, and has the advantages of low fluorescence background, adjustable melting temperature, good tolerance, low cost, simple operation, less contamination, high sensitivity, wide application range, etc.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to the following patent application filed on December 27, 2021: 1. A Chinese patent application entitled "Primer probe composition, detection method and detection apparatus" with application number CN202111614026.1. This application also claims priority to the following patent application filed on December 2, 2022: 2. A Chinese patent application entitled "Primer probe composition and detection method" with application number CN202211539752.6. This application also claims priority to the following patent application filed on October 18, 2022: 3. A Chinese patent application entitled "Multiple time-division nucleic acid detection apparatus and detection method" with application number CN202211271772.X. The contents of the foregoing are hereby incorporated by reference to the extent consistent with the present invention.

[0002] [Technical field] The present invention relates to the field of nucleic acid sequence detection, in particular to a method for detecting target nucleic acids, and more particularly to a method for detecting single or multiplex target nucleic acids in digital PCR using a universal probe. [Background technology]

[0003] Polymerase chain reaction (PCR) is a molecular biology technique that enzymatically replicates DNA without the use of living organisms. PCR is commonly used in medical and biological research laboratories for a variety of tasks, such as infectious disease diagnosis, gene cloning, laboratory animal phenotyping, transcriptome research, genetic disease detection, genetic fingerprinting, parentage testing, etc. Due to its unparalleled replication ability and accuracy, PCR is recognized by molecular biologists as the preferred method for nucleic acid detection. In the late 1990s of the last century, the real-time fluorescent quantitative PCR (qPCR) technology and related products provided by ABI, USA, developed PCR into a highly sensitive, highly specific and accurately quantitative nucleic acid sequence analysis technology.

[0004] Digital PCR (dPCR) technology is a nucleic acid molecule absolute quantification technology, which uses the principle of limiting dilution to distribute one fluorescent quantitative PCR reaction system into thousands to tens of thousands of single nanoliter-level microreactors, each of which contains or does not contain one or multiple copies of the target nucleic acid molecule (DNA target), and simultaneously performs single molecule template PCR amplification. Unlike fluorescent quantitative PCR, which collects fluorescence during each amplification cycle, digital PCR collects the fluorescent signal of each reaction cell independently after the amplification is completed, and finally obtains the original copy number or concentration of the target molecule according to the principle of Poisson distribution and the proportion of positive / negative reaction cells.

[0005] Compared with fluorescent quantitative PCR, digital PCR can perform accurate absolute quantitative detection without relying on Ct value and calibration curve, and has the advantages of high sensitivity and high accuracy. Digital PCR only judges two amplification states, "yes" and "no", when determining the result, so there is no need to detect the intersection of the fluorescent signal with the set threshold line, and it does not depend on the identification of Ct value at all, so the influence of amplification efficiency on the digital PCR reaction and result judgment is greatly reduced, and the resistance to PCR reaction inhibitors is greatly improved. In addition, the process of distributing in the reaction system in digital PCR experiments can locally greatly reduce the concentration of background sequences that have a competitive effect with the target sequence, so digital PCR is particularly suitable for detecting rare mutations in complex backgrounds, and is currently widely applied in liquid biopsy to realize the detection of rare mutation markers in the peripheral blood of tumor patients.

[0006] In the process of multiplexed nucleic acid detection, conventional sample detection devices first amplify nucleic acids containing the target sequences to be detected, and then collect signals at different temperatures. There may be cases where the signals acquired at different temperatures overlap (i.e., different target sequences generate signals simultaneously). Conventional calculation methods use image processing or other signal calculation methods to separate the overlapping signals and calculate the signal values ​​generated by each target sequence, but such methods are complex and prone to calculation errors. Summary of the Invention

[0007] In order to solve the above problems, the present invention provides a method for detecting target nucleic acid, which can detect one target or two or more target nucleic acids, and the method first amplifies a nucleic acid containing a target sequence to be detected, and then performs signal collection, and a single signal can be obtained at different temperatures by setting the annealing temperature during amplification and the collection temperature during signal collection (only one target sequence at most generates a signal during one signal collection).When there are two or more targets, the problem to be solved by the present invention also includes how to detect and distinguish multiple different targets in a single tube reaction, and how to avoid the generation of false positive signals caused by primer dimers when multiple pairs of primers are present.

[0008] Therefore, in the first aspect, the present invention provides a method for detecting a target nucleic acid, comprising: Step S100 of mixing a primer-probe composition, a test sample, and an amplification reagent containing a DNA polymerase and dNTPs to obtain a reaction system; Step S200 of performing a first round of PCR amplification on the reaction system, wherein the annealing temperature used during amplification is T1; Step S300 of performing any of the following operations: Operation 1 including S310 of performing a first signal collection at a first signal collection temperature t1; S320 of performing a second round of PCR amplification, wherein the annealing temperature used during amplification is T2; Operation 2 including S310' of performing a first signal collection at a first signal collection temperature t1; S310'' of performing a second round of PCR amplification, wherein the annealing temperature used during amplification is T2; Operation 3 including S320'' of performing a first signal collection at a first signal collection temperature t1 and S320'' of performing a second signal collection at a second signal collection temperature t2; The above operations satisfy T1>T2 and T2<t1; Step S400 of performing a second signal collection at a second signal collection temperature t2, where t2>t1 and t2>T1; Here, the type of the target to be detected in the test sample is one; In the first signal collection and the second signal collection, a method for detecting a target nucleic acid is provided, wherein the target signal is included in at most one signal collection.

[0009] In some embodiments, after the first PCR amplification or after the first and second PCR amplifications, signal acquisition is performed once, and the signal acquisition temperature increases sequentially (t2>t1). In some embodiments, whether PCR is performed twice continuously or only once, double-stranded products bound to the probe can be generated. When signals are acquired at a relatively low temperature (t1), due to the formation of double-stranded products, a detectable change in the signal occurs. Subsequently, when signals are acquired at a higher temperature (t2), since t2 is higher than the melting temperature of the double-stranded product, the double-stranded product becomes single-stranded, and the signal acquired at t2 is different from the signal acquired at t1, that is, the signals acquired twice are different, and the presence of the target nucleic acid can be more accurately verified.

[0010] In some embodiments, two PCRs and two signal acquisitions are performed, and PCR and signal acquisition are performed alternately. The annealing temperature employed for PCR amplification decreases sequentially (T2<T1), while the signal acquisition temperature increases sequentially (t2>t1). That is, when PCR cycle amplification is performed using a certain temperature as the annealing temperature of PCR, the annealing temperature of the single-stranded pre-product formed by the target nucleic acid is lower than this temperature, and it cannot bind to the probe to form a double-stranded product, and no change in the detection signal occurs. Immediately after this PCR amplification, when signals are acquired at another temperature, the double-stranded product of the single-stranded pre-product formed by the target nucleic acid and the probe becomes double-stranded, generating a detectable target nucleic acid signal. The signals collected twice are different, more accurately verifying the presence of the target nucleic acid.

[0011] Therefore, by adopting the above PCR detection method, the presence or absence of the target nucleic acid can be more accurately verified. When the target nucleic acid is present, the signals collected twice are different, and in only one signal acquisition, at most the signals of one type of target nucleic acid are included. When the target nucleic acid is absent, no target nucleic acid signal is present in either of the two signal acquisitions.

[0012] T2 < T1 means that the annealing temperature of the second PCR has a difference in magnitude from the annealing temperature of the first PCR. Due to the difference in the annealing temperatures of the two, after the second PCR amplification, the number of types of double-stranded products formed with a certain probe is at most one more than the number of types of double-stranded products formed with that probe after the first PCR amplification (not increasing means that there is no corresponding target nucleic acid in the test product). In some embodiments, the temperature difference between T2 and T1 is 4 °C or more, for example, the difference is 4 - 40 °C, and also, for example, the difference is 4 - 30 °C, 4 - 20 °C, 4 - 18 °C, 4 - 15 °C, 4 - 12 °C, 4 - 10 °C, 4 - 8 °C, 4 - 6 °C or 4 - 5 °C, etc.

[0013] t2 > t1 means that the temperature of the second signal acquisition has a difference in magnitude from the temperature of the first signal acquisition. Due to the difference in the two temperatures, the signal of the second signal acquisition is different from the signal of the first signal acquisition. In some embodiments, the temperature difference between t2 and t1 is 6 °C or more, for example, the difference is 6 - 46 °C, and also, for example, the difference is 6 - 40 °C, 6 - 35 °C, 6 - 30 °C, 6 - 25 °C, 6 - 20 °C, 6 - 18 °C, 6 - 15 °C, 6 - 12 °C, 6 - 10 °C, 6 - 8 °C or 6 - 7 °C, etc.

[0014] T2 < t1 means that the annealing temperature of the second PCR has a difference in magnitude from the temperature of the first signal acquisition. In some embodiments, the temperature difference between T2 and t1 is 5 °C or more, for example, the difference is 5 - 50 °C, and also, for example, the difference is 5 - 40 °C, 5 - 30 °C, 5 - 20 °C, 5 - 18 °C, 5 - 15 °C, 5 - 12 °C, 5 - 10 °C, 5 - 8 °C, 5 - 7 °C or 5 - 6 °C, etc.

[0015] t2 > T1 means that the annealing temperature of the first PCR has a difference in magnitude from the temperature of the second signal acquisition. In some embodiments, the temperature difference between t2 and T1 is 7 °C or more, for example, the difference is 7 - 50 °C, and also, for example, the difference is 7 - 40 °C, 7 - 30 °C, 7 - 20 °C, 7 - 18 °C, 7 - 15 °C, 7 - 12 °C, 7 - 10 °C or 7 - 8 °C, etc.

[0016] In some embodiments of the present invention, in the above detection method, the annealing temperature used during the first PCR amplification is T1, the collection temperature used during the first signal collection is t1, and T1 ≤ t1.

[0017] In some embodiments, during the first PCR amplification (the annealing temperature thereof is T1), among the single-stranded pre-products generated by the primer set and the probe, one type of single-stranded pre-product specifically binds to the probe and forms a double-stranded product after 0 base extensions, and the annealing temperature of the single-stranded pre-product is slightly lower than its melting temperature (in the normal case, the annealing temperature of an oligonucleotide is usually lower than the melting temperature (Tm) of the oligonucleotide). Signal collection is performed at t1 (T1 ≤ t1 < its melting temperature), and at this time, the single-stranded pre-product and the probe exhibit a double-stranded state, causing a detectable signal change in the probe. In some embodiments, signal collection may also be performed at t1 (t1 ≤ T1), and at this time as well, the single-stranded pre-product and the probe exhibit a double-stranded state, causing a detectable signal change in the probe. Those skilled in the art can specifically select the temperature of the signal collection temperature t1 according to actual needs (for example, actual needs such as when signal collection is performed, the single-stranded pre-product and the probe are in a double-stranded state and the collected signal intensity is large).

[0018] According to a preferred embodiment of the present invention, the annealing temperature range is 40°C - 75°C, that is, 40°C ≤ T2 < T1 ≤ 75°C.

[0019] According to a preferred embodiment of the present invention, the signal collection temperature is 0 - 95°C, that is, 0°C ≤ t1 < t2 ≤ 90°C. Usually, the selection of the signal collection temperature can be determined by those skilled in the art according to the actual situation of the designed primer-probe composition. For example, due to the design of the primer-probe composition, those skilled in the art can predict that when the melting temperature of the reaction product representing the target nucleic acid is T and it is desired to detect the signal of the target nucleic acid, the signal collection temperature ≤ T; when it is not desired to detect the signal of the target nucleic acid, the signal collection temperature > T.

[0020] According to a preferred embodiment of the present invention, the reaction conditions for the first PCR amplification in step S200 include pre-denaturation at about 85°C-about 105°C for about 0-about 15 minutes, denaturation at about 85°C-about 105°C for about 1-about 60 seconds, annealing and extension at about 40°C-about 75°C for about 3-about 90 seconds, and 20-60 cycles. Preferably, when the target in the test sample is RNA, the amplification reagent further includes a reverse transcriptase, and the reaction conditions for the first PCR amplification in the reaction system include reverse transcription at about 30°C-about 65°C for about 2-about 30 minutes, pre-denaturation at about 85°C-about 105°C for about 0-about 15 minutes, denaturation at about 85°C-about 105°C for about 1-about 60 seconds, annealing and extension at about 40°C-about 75°C for about 3-about 90 seconds, and 20-60 cycles. And / or, the second PCR amplification in step S300 includes reaction conditions of denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 3 to about 90 seconds, and 1 to 20 cycles, or incubation at a constant temperature of about 20°C to about 75°C for about 10 to about 1800 seconds.

[0021] According to a preferred embodiment of the present invention, the reaction conditions for the first PCR amplification in step S200 include pre-denaturation at about 90°C-about 96°C for about 5-about 15 minutes, denaturation at about 90°C-about 95°C for about 10-about 60 seconds, annealing and extension at about 50°C-about 75°C for about 30-about 90 seconds, and 35-50 cycles. More preferably, when the target in the test sample is RNA, the amplification reagent further includes a reverse transcriptase, and the reaction conditions for the first PCR amplification in the reaction system include reverse transcription at about 30-about 65°C for about 2-about 30 minutes, pre-denaturation at about 90°C-about 96°C for about 5-about 15 minutes, denaturation at about 90°C-about 95°C for about 10-about 60 seconds, annealing and extension at about 50°C-about 75°C for about 30-about 90 seconds, and 35-50 cycles. And / or, the second PCR amplification in step S300 includes reaction conditions of denaturation at about 85°C to about 105°C for about 2 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 10 to about 90 seconds, and 1 to 20 cycles, or incubation at a constant temperature of about 20°C to about 75°C for about 10 to about 1800 seconds.

[0022] According to a preferred embodiment of the present invention, before performing a first PCR amplification on the reaction system, the method further includes distributing the reaction system into 500 or more reaction cells, such that each reaction cell contains one target nucleic acid of a test sample or does not contain any target nucleic acid of a test sample, and more preferably, the signal collection is collecting a fluorescent signal by a camera, and the signal channel collection is collecting a fluorescent signal by a camera in a fluorescent signal channel.

[0023] According to a preferred embodiment of the present invention, the primer probe composition comprises a probe (P) and a primer, the primer specifically binds to a target in the test sample and then generates a pre-product, the pre-product comprises one single-stranded pre-product capable of specifically binding to a probe, the single-stranded pre-product specifically binds to the probe and elongates by ≧0 bases to form a double-stranded product, and the formation of the double-stranded product causes a change in the probe signal, more preferably, the elongation is an elongation of 0-100 bases, even more preferably, the elongation is an elongation of 1-100 bases, and the upper limit of the number of extended bases is the full length of the probe, and / or the concentration of the primer is about 30nM-about 1000nM, and the concentration of the probe (P) is about 100nM-about 1200nM. In the present invention, the concentration unit nM means nmol / L.

[0024] Specifically, the extension of 0 bases means that the single-stranded pre-product is not extended after specifically binding (hybridizing) with the probe (P). Those skilled in the art can achieve the effect of "specifically binding but not extending" by designing the primer sequence in the primer set and / or the sequence of the probe according to actual needs, for example, by designing the generated first single-stranded pre-product to bind to the 5' end of the probe, or by designing the generated first single-stranded pre-product to include a region at the 3' end that is not complementary to the probe.

[0025] According to a preferred embodiment of the present invention, the probe (P) is a freely designed sequence that does not pair with any target sequence, includes a probe signal detection region (H), and is modified with a detection label; the primer includes a forward primer (F) and / or a reverse primer (R); The forward primer (F) comprises a target sequence binding region that specifically binds to a target sequence, the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region, or the reverse primer (R) comprises an extension block region (M), a primer signal detection region (h) and a target sequence binding region in this order from the 5' end to the 3' end; wherein the target sequence binding region of the reverse primer (R) is a sequence that specifically binds to a target sequence, the primer signal detection region (h) of the reverse primer (R) is a sequence that does not bind to any target sequence, the complementary sequence (h') of the primer signal detection region (h) specifically binds to the probe signal detection region (H) of the probe (P), and the extension block region (M) of the reverse primer (R) is a freely designed sequence that is neither identical to nor complementary to any part of the sequence of the probe (P) or any target sequence, More preferably, there is an interval of 0-20 bases between the primer signal detection region (h) and the target sequence binding region in the reverse primer (R). Here, the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them. In some embodiments, there is an interval of 0-20 bases between the extension block region (M) and the primer signal detection region (h) in the reverse primer (R). Here, the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them.

[0026] In the present invention, "specifically pairing" or "specific binding" refers to two single-stranded nucleic acid molecules having complementary sequences pairing their bases complementarily under certain conditions (such as appropriate temperature and ionic strength). As is well known to those skilled in the art, two single-stranded nucleic acid molecules do not necessarily have to be completely complementary to each other to specifically bind, and can also specifically bind even when there are a small number of base mismatches.

[0027] According to a preferred embodiment of the present invention, the forward primer (F) further comprises a probe anchor region (A), and the forward primer (F) comprises the probe anchor region (A) and a target sequence binding region in this order from the 5' end to the 3' end, wherein the probe anchor region (A) is a sequence that does not bind to any target sequence, and more preferably, there is an interval of 0-20 bases between the probe anchor region (A) and the target sequence binding region, wherein the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by these, The probe (P) further comprises a primer anchor region (A'), which has a sequence complementary to the probe anchor region (A) of the forward primer (F), and more preferably, there is a gap of 0-20 bases between the primer anchor region (A') and the probe signal detection region (H) in the probe (P).

[0028] According to a preferred embodiment of the present invention, the structures of the forward primer and the reverse primer are interchangeable. For example, the primer structure of "the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the forward primer (F) comprises a target sequence binding region" can be interchangeable with "the forward primer (F) comprises a primer signal detection region (h) and a target sequence binding region, and the reverse primer (R) comprises a target sequence binding region", or the primer structure of "the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the forward primer (F) comprises a probe anchor region (A) and a target sequence binding region" can be interchangeable with "the forward primer (F) comprises a primer signal detection region (h) and a target sequence binding region, and the reverse primer (R) comprises a probe anchor region (A) and a target sequence binding region".

[0029] It is well known to those skilled in the art that in order to avoid primer dimers, the target sequence binding region in the forward primer (F) is different from and cannot specifically bind to the target sequence binding region in the reverse primer (R).

[0030] According to a preferred embodiment of the present invention, in the primer, at least one of the forward primers (F) comprises a first forward primer and a second forward primer, the first forward primer comprises a forward amplification product capture region (B) and a target sequence binding region in the order from the 5' end to the 3' end, and the second forward primer comprises a forward amplification product capture region (B), and the forward amplification product capture region (B) does not pair with and bind to the target sequence or any fragment thereof. More preferably, there is an interval of 0-20 bases between the forward amplification product capture region (B) and the target sequence binding region in the first forward primer.

[0031] According to a preferred embodiment of the present invention, the second forward primer further comprises a probe anchor region (A), and more preferably, there is a 0-20 base interval between the probe anchor region (A) and the forward amplification product capture region (B) in the second forward primer.

[0032] According to a preferred embodiment of the present invention, in the primer, at least one of the reverse primers (R) includes a first reverse primer and a second reverse primer, the first reverse primer includes a reverse amplification product capture region (C) and a target sequence binding region from the 5' end to the 3' end in this order, and the second reverse primer includes a primer signal detection region (h) and a reverse amplification product capture region (C) from the 5' end to the 3' end in this order, the reverse amplification product capture region (C) does not pair with and bind to a target sequence or any fragment thereof, More preferably, there is a 0-20 base interval between the reverse amplification product capture region (C) and the target sequence binding region in the first reverse primer, and / or there is a 0-20 base interval between the primer signal detection region (h) and the reverse amplification product capture region (C) in the second reverse primer, where the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them.

[0033] In some embodiments of the present invention, the method for detecting the target nucleic acid is a digital PCR detection method.In some embodiments of the present invention, the method for detecting the target nucleic acid further comprises distributing the reaction system into 500 or more reaction cells before carrying out a first PCR amplification on the reaction system, so that each reaction cell contains one target nucleic acid of a test sample or does not contain a target nucleic acid of a test sample, and preferably, the signal collection is collecting a fluorescent signal by a camera, and the signal channel collection is collecting a fluorescent signal by a camera in a fluorescent signal channel.In the method for detecting the target nucleic acid, the primer probe composition may be a conventional primer probe composition in the art, for example, a primer probe composition containing a molecular beacon, or a primer probe composition for detecting a nucleic acid sequence provided in the third aspect below.

[0034] Therefore, in a second aspect, the present invention provides a method for producing a method for treating a pulmonary arthritis, comprising: A step of mixing a primer probe composition containing at least one probe (P) and at least two primers for different targets, a test sample, and an amplification reagent containing DNA polymerase and dNTPs to obtain a reaction system; and performing n PCR amplifications and n signal collections on the reaction system, wherein one signal collection is performed after each PCR amplification, each signal collection includes at least one signal channel collection, and each signal channel collection includes at most one target signal; The annealing temperature used during the n-th PCR amplification is Tn, the acquisition temperature used during the n-th signal acquisition is tn, Tn < tn, Tn < T(n - 1), and Tn < t(n - 1), tn > t(n - 1), and tn > T(n - 1). Provided is a method for detecting two or more target nucleic acids, where n is an integer greater than or equal to 2, and n is less than or equal to the number of types of target substances in the test sample.

[0035] In some embodiments, signal acquisition is performed once after each PCR amplification. The annealing temperature used for PCR amplification decreases sequentially (Tn < T(n - 1)), while the signal acquisition temperature increases sequentially (tn > t(n - 1)). That is, when performing PCR cycle amplification with a certain temperature as the annealing temperature of PCR, single-stranded pre-products with an annealing temperature higher than this temperature can all bind to the probe to form double-stranded products and cause changes in the detection signal. Single-stranded pre-products with an annealing temperature lower than this temperature remain in a single-stranded state. Immediately after this PCR amplification, signal acquisition is performed at another temperature, and only one type of single-stranded pre-product forms a double-stranded product with the probe and enters a double-stranded state, generating a detectable target nucleic acid signal. (For other types of single-stranded pre-products, some cannot form double-stranded products by annealing with the probe during the PCR cycle amplification stage because their annealing temperature is lower, and some can form double-stranded products by annealing with the probe during the PCR cycle amplification stage because their annealing temperature is higher. However, the melting temperature of these double-stranded products is lower and lower than the signal acquisition temperature, so the generated double-stranded products also exhibit a single-stranded state at this signal acquisition temperature.) Therefore, according to the above PCR detection method, there is at most one type of target nucleic acid signal in the signal collected from each signal channel. If the target nucleic acid does not exist, it can be realized that there is no signal of the target nucleic acid.

[0036] Tn < T(n - 1) means that the annealing temperature of the n-th PCR has a difference in magnitude from the annealing temperature of the (n - 1)-th PCR. Due to the difference in the annealing temperatures of the two, after the n-th PCR amplification, the number of types of double-stranded products formed with a certain probe is at most one more than the number of types of double-stranded products formed with that type of probe after the (n - 1)-th PCR amplification (not increasing means that there is no corresponding target nucleic acid in the test product). In some embodiments, the temperature difference between Tn and T(n - 1) is 4°C or more, for example, the difference is 4 - 40°C, and also, for example, the difference is 4 - 30°C, 4 - 20°C, 4 - 18°C, 4 - 15°C, 4 - 12°C, 4 - 10°C, 4 - 8°C, 4 - 6°C or 4 - 5°C, etc.

[0037] tn > t(n - 1) means that the temperature of the n-th signal acquisition has a difference in magnitude from the temperature of the (n - 1)-th signal acquisition. Due to the difference between the two temperatures, the double-stranded product during the n-th signal acquisition is different from the double-stranded product during the (n - 1)-th signal acquisition. For example, the double-stranded product during the (n - 1)-th signal acquisition is A, and the double-stranded product during the n-th signal acquisition is B (at the time of the n-th signal acquisition, since the melting temperature of double-stranded product A is lower than the temperature at the time of the n-th signal acquisition, at this time, double-stranded product A is in a single-stranded state). In some embodiments, the temperature difference between tn and t(n - 1) is 6°C or more, for example, the difference is 6 - 46°C, and also, for example, the difference is 6 - 40°C, 6 - 35°C, 6 - 30°C, 6 - 25°C, 6 - 20°C, 6 - 18°C, 6 - 15°C, 6 - 12°C, 6 - 10°C, 6 - 8°C or 6 - 7°C, etc.

[0038] Tn < tn means that the annealing temperature of the n-th PCR has a difference in magnitude from the temperature of the n-th signal acquisition. Due to the difference between the two temperatures, a specific double-stranded product can be obtained after the n-th PCR amplification, and the signal of the double-stranded product can be obtained during the n-th signal acquisition. In order to obtain the signal of the double-stranded product, during the n-th signal acquisition (under the condition of the signal acquisition temperature tn), the double-stranded product does not show a single-stranded state. In some embodiments, the difference between Tn and tn is 1 °C or more. For example, the difference is 1 - 50 °C, 1 - 40 °C, 1 - 30 °C, 1 - 25 °C, 2 - 20 °C, 2 - 15 °C, 2 - 12 °C, 2 - 10 °C, 2 - 7 °C, 2 - 6 °C, 2 - 5 °C, 3 - 5 °C or 4 - 5 °C, etc. In some embodiments, the signal acquisition temperature tn used is below the melting temperature of the double-stranded product.

[0039] Tn < t(n - 1) means that the annealing temperature of the n-th PCR has a difference in magnitude from the temperature of the (n - 1)-th signal acquisition. Due to the difference between the two temperatures, the double-stranded product during the (n - 1)-th signal acquisition still exists during the n-th PCR, and at the same time, another double-stranded product different from the double-stranded product is generated. In some embodiments, the temperature difference between Tn and t(n - 1) is 5 °C or more. For example, the difference is 5 - 50 °C. Also, for example, the difference is 5 - 40 °C, 5 - 30 °C, 5 - 20 °C, 5 - 18 °C, 5 - 15 °C, 5 - 12 °C, 5 - 10 °C, 5 - 8 °C, 5 - 7 °C or 5 - 6 °C, etc.

[0040] tn > T(n - 1) means that the annealing temperature of the (n - 1)-th PCR has a difference in magnitude from the temperature of the n-th signal acquisition. In some embodiments, the temperature difference between tn and T(n - 1) is 7 °C or more. For example, the difference is 7 - 50 °C. Also, for example, the difference is 7 - 40 °C, 7 - 30 °C, 7 - 20 °C, 7 - 18 °C, 7 - 15 °C, 7 - 12 °C, 7 - 10 °C or 7 - 8 °C, etc.

[0041] In the present invention, the number of types of probes is classified according to the number of types of their detection labels. Different types of probes have different detection labels modified therein (in some embodiments, the detection labels are detection groups), and can achieve signal collection in different signal channels (or fluorescence channels). For example, the primer probe composition of the present invention includes two types of probes, the detection groups modified on one probe are VIC and BHQ1, and the double-stranded product formed by specific binding with this type of probe is signal collected in the VIC channel, and the detection groups modified on the other probe are FAM and BHQ1, and the double-stranded product formed by specific binding with this type of probe is signal collected in the FAM channel.

[0042] In some embodiments, one signal collection is performed after each PCR amplification, and each signal collection includes m signal channel collections, m being an integer ≧1, and m being the number of types of detection groups of the probe in the primer probe composition. As described above, after each PCR amplification, the number of types of double-stranded products formed with the same probe increases by at most one type compared to the previous PCR amplification. In the primer probe composition, if the number of types of probes is m, after each PCR amplification, the number of types of double-stranded products formed with m types of probes increases by at most m types compared to the previous PCR amplification (the number of types of double-stranded products formed per one type of probe increases by at most one type). By collecting m different signal channels, the target nucleic acid signals corresponding to the increased m types of double-stranded products can be distinguished and do not cross each other, thereby realizing multiplex detection.

[0043] According to a preferred embodiment of the present invention, the at least one signal collection includes m signal channel collections, where m is the number of types of detection labels of the probes in the primer probe composition, and the product of n and m is ≧ the number of types of targets to be detected in the test sample. In order to facilitate the program design of the detection device, in some embodiments, when there are two types of probes in the primer probe composition, each signal collection is performed twice in two signal channels. In some embodiments, when there are five types of targets in the test sample, and the primer probe composition for these five types of targets contains two types of probes, the types of targets detected by the first probe are three types, and the types of targets detected by the second probe are two types, three signal collections are performed on the reaction product, and each signal collection is two signal channel collections performed in two signal channels. In this way, in a certain signal channel, three signal collections are performed, but there are only two types of corresponding targets, and there is no target in the signal of a certain signal channel collection, or the signals of the adjacent two signal channel collections are the same. In these embodiments, the detection channel with the largest number of targets is used as a reference to set the number of signal collections, so as to realize that the number of signal collections is less than the number of types of target nucleic acid, and the number of signal collections is reduced. Although it does not completely satisfy the requirement that the number of signal collections of other signal channels is the smallest, the setting of the instrument program is more convenient, and those skilled in the art can select according to actual needs. In some embodiments, those skilled in the art can select not to collect signals in channels without desired targets according to the design situation of the primer probe composition, thereby reducing the number of signal collections and simplifying the operation steps.

[0044] In some embodiments of the present invention, in the above detection method, the annealing temperature used in the first PCR amplification is T1, the collection temperature used in the first signal collection is t1, and T1≦t1.

[0045] In some embodiments, during the first PCR amplification (the annealing temperature thereof is T1), among the single-stranded pre-products generated by the primer set and the probe, one type of single-stranded pre-product specifically binds to the probe and forms a double-stranded product after 0 base extensions, and the annealing temperature of the single-stranded pre-product is slightly lower than its melting temperature (in the normal case, the annealing temperature of an oligonucleotide is usually lower than the melting temperature (Tm) of the oligonucleotide). Signal collection is performed at t1 (T1≤t1<its melting temperature), and at this time, the single-stranded pre-product and the probe present a double-stranded state, causing a detectable signal change in the probe. In some embodiments, signal collection may be performed at t1 (t1≤T1), and at this time, the single-stranded pre-product and the probe also present a double-stranded state, causing a detectable signal change in the probe. Those skilled in the art can specifically select the temperature of the signal collection temperature t1 according to the actual needs (for example, the actual needs such as the single-stranded pre-product and the probe being in a double-stranded state and the collected signal intensity being large during signal collection).

[0046] In some embodiments, the annealing temperature is 40°C≤Tn<T(n - 1)≤75°C (n is an integer ≥2).

[0047] In some embodiments, the sampling temperature is 0°C≤t(n - 1)<tn≤90°C (n is an integer ≥2).

[0048] According to a preferred embodiment of the present invention, the reaction conditions for the first PCR amplification of the reaction system include pre-denaturation at about 85°C-about 105°C for about 0-about 15 minutes, denaturation at about 85°C-about 105°C for about 1-about 60 seconds, annealing and extension at about 40°C-about 75°C for about 3-about 90 seconds, and 20-60 cycles. Preferably, when the target in the test sample is RNA, the amplification reagent further includes a reverse transcriptase, and the reaction conditions for the first PCR amplification of the reaction system include reverse transcription at about 30-about 65°C for about 2-about 30 minutes, pre-denaturation at about 85°C-about 105°C for about 0-about 15 minutes, denaturation at about 85°C-about 105°C for about 1-about 60 seconds, annealing and extension at about 40°C-about 75°C for about 3-about 90 seconds, and 20-60 cycles.

[0049] According to a preferred embodiment of the present invention, the reaction conditions for the first PCR amplification of the reaction system include pre-denaturation at about 90°C-about 96°C for about 5-about 15 minutes, denaturation at about 90°C-about 95°C for about 10-about 60 seconds, annealing and extension at about 50°C-about 75°C for about 30-about 90 seconds, and 35-50 cycles. Preferably, when the target in the test sample is RNA, the amplification reagent further includes a reverse transcriptase, and the reaction conditions for the first PCR amplification of the reaction system include reverse transcription at about 30-about 65°C for about 2-about 30 minutes, pre-denaturation at about 90°C-about 96°C for about 5-about 15 minutes, denaturation at about 90°C-about 95°C for about 10-about 60 seconds, annealing and extension at about 50°C-about 75°C for about 30-about 90 seconds, and 35-50 cycles.

[0050] According to a preferred embodiment of the present invention, the reaction conditions for the first PCR amplification of the reaction system are as follows: pre-denaturation at about 85° C.-about 105° C. for about 0-about 15 minutes, denaturation at about 85° C.-about 105° C. for about 1-about 60 seconds, annealing and extension at about 40° C.-about 75° C. for about 3-about 90 seconds, and about 20-about 60 cycles of denaturation at about 85° C.-about 105° C. for about 1-about 60 seconds, annealing and extension at about 20° C.-about 75° C. for about 3-about 90 seconds. or the reaction conditions for performing a first PCR amplification on the reaction system include pre-denaturing at about 85° C. to about 105° C. for about 0 to about 15 minutes, denaturing at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and elongating at about 40° C. to about 75° C. for about 3 to about 90 seconds, performing 20 to 60 cycles, and incubating at a constant temperature of about 20° C. to about 75° C. for about 10 to about 1800 seconds; Preferably, when the target in the test sample is RNA, the amplification reagent further comprises a reverse transcriptase, and the reaction conditions for the first PCR amplification in the reaction system are reverse transcription at about 30 to about 65° C. for about 2 to about 30 minutes, pre-denaturation at about 85° C. to about 105° C. for about 0 to about 15 minutes, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 40° C. to about 75° C. for about 3 to about 90 seconds, 20 to 60 cycles, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, and annealing and extension at about 20° C. to about 75° C. for about 1 to about 60 seconds. or reaction conditions for performing a first PCR amplification on the reaction system include reverse transcription at about 30°C to about 65°C for about 2 to about 30 minutes, pre-denaturation at about 85°C to about 105°C for about 0 to about 15 minutes, denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 40°C to about 75°C for about 3 to about 90 seconds, 20 to 60 cycles, and incubation at a constant temperature of about 20°C to about 75°C for about 10 to about 1800 seconds.

[0051] According to a preferred embodiment of the present invention, the reaction conditions for the first PCR amplification of the reaction system are as follows: pre-denaturation at about 90° C.-about 96° C. for about 5-about 15 minutes, denaturation at about 90° C.-about 95° C. for about 10-about 60 seconds, annealing and extension at about 50° C.-about 75° C. for about 30-about 90 seconds, and about 35-about 50 cycles of denaturation at about 90° C.-about 95° C. for about 10-about 60 seconds, annealing and extension at about 20° C.-about 75° C. for about 30-about 90 seconds. or the reaction conditions for performing a first PCR amplification on the reaction system include pre-denaturing at about 90° C. to about 96° C. for about 5 to about 15 minutes, denaturing at about 90° C. to about 95° C. for about 10 to about 60 seconds, annealing and elongating at about 50° C. to about 75° C. for about 30 to about 90 seconds, performing 35 to 50 cycles, and incubating at a constant temperature of about 20° C. to about 75° C. for about 10 to about 1000 seconds; Preferably, when the target in the test sample is RNA, the amplification reagent further comprises a reverse transcriptase, and the reaction conditions for the first PCR amplification in the reaction system are reverse transcription at about 30 to about 65° C. for about 2 to about 30 minutes, pre-denaturation at about 90° C. to about 96° C. for about 5 to about 15 minutes, denaturation at about 90° C. to about 95° C. for about 10 to about 60 seconds, annealing and extension at about 50° C. to about 75° C. for about 30 to about 90 seconds, 35 to 50 cycles, denaturation at about 90° C. to about 95° C. for about 10 to about 60 seconds, and annealing and extension at about 20° C. to about 75° C. or the reaction conditions for performing a first PCR amplification on the reaction system include reverse transcription at about 30 to about 65°C for about 2 to about 30 minutes, pre-denaturation at about 90°C to about 96°C for about 5 to about 15 minutes, denaturation at about 90°C to about 95°C for about 10 to about 60 seconds, annealing and extension at about 50°C to about 75°C for about 30 to about 90 seconds, 35 to 50 cycles, and incubation at a constant temperature of about 20°C to about 75°C for about 10 to about 1000 seconds.

[0052] According to a preferred embodiment of the present invention, the reaction conditions for the other PCR amplifications after the first PCR amplification include denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 3 to about 90 seconds, and 1 to 20 cycles, or incubation at a constant temperature of about 20°C to about 75°C for 10 to 1800 seconds.

[0053] According to a preferred embodiment of the present invention, the reaction conditions for the other PCR amplifications after the first PCR amplification include denaturation at about 90°C to about 95°C for about 10 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 30 to about 90 seconds, and 1 to 20 cycles, or incubation at a constant temperature of about 20°C to about 75°C for 10 to 1000 seconds.

[0054] According to a preferred embodiment of the present invention, at least one of the primers generates a pre-product after specifically binding to a target, the pre-product includes one single-stranded pre-product capable of specifically binding to a probe (P), the single-stranded pre-product forms a double-stranded product after specifically binding to the probe (P) and being extended, the formation of the double-stranded product causes a change in the probe signal, preferably, the extension is an extension of 0-100 bases, more preferably an extension of 1-100 bases, the upper limit of the number of extended bases is the full length of the probe, and / or the concentration of the primer is about 30 nM-about 1000 nM and the concentration of the probe (P) is about 100 nM-about 1200 nM. When multiple targets share a single probe, the amount of probe used can be higher, for example, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, 2000 nM or more.

[0055] Specifically, the extension of 0 bases means that the single-stranded pre-product is not extended after specifically binding (hybridizing) with the probe (P). Those skilled in the art can achieve the above-mentioned effect of "specifically binding but not extending" by designing the primer sequence in the primer set and / or the sequence of the probe according to actual needs, for example, by designing the generated first single-stranded pre-product to bind to the 5' end of the probe, or by designing the generated first single-stranded pre-product to include a region at the 3' end that does not complementarily pair with the probe.

[0056] According to a preferred embodiment of the present invention, the at least two primers for different targets include any one of the following: (1) at least two types of forward primers (F) and at least one type of reverse primer (R) (2) at least one forward primer (F) and at least two reverse primers (R); (3) at least two forward primers (F) and at least two reverse primers (R); The different forward primers (F) each independently contain a target sequence binding region that specifically binds to a different target sequence, and / or, the probe (P) is a freely designed sequence that does not bind to any target sequence, includes a probe signal detection region (H), and is modified with a detection label; And / or, the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region, wherein the target sequence binding regions of the different reverse primers (R) have sequences that specifically bind to different target sequences, and the primer signal detection regions (h) of the different reverse primers (R) do not bind to any target sequence but have a sequence that is partially or entirely the same as the probe signal detection region (H) of the probe (P), and preferably the sequences of the primer signal detection regions (h) of the different reverse primers (R) are different from each other.

[0057] In the present invention, the primer signal detection region (h) of the reverse primer (R) having a sequence which is partially or entirely the same as the probe signal detection region (H) of the probe (P) means that the complementary sequence of the primer signal detection region (h) of the reverse primer (R) specifically pairs and binds to the probe signal detection region (H) of the probe (P).

[0058] According to a preferred embodiment of the present invention, when the primer comprises at least two kinds of the primer, the forward primer and the reverse primer have exchangeable structures, so the ratio of the forward primer and the reverse primer is not limited.In some embodiments, the content of the primer that does not include the primer signal detection region h is more than the primer that includes the other primer signal detection region h, preferably, the content of the primer that does not include the primer signal detection region h is 1.2-20 times the content of the primer that includes the other primer signal detection region h, more preferably, the content of the primer that does not include the primer signal detection region h is 2 times, 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times the content of the primer that includes the other primer signal detection region h.

[0059] According to a preferred embodiment of the present invention, in the primer, at least one type of the reverse primer (R) comprises, from the 5' end to the 3' end, an extension block region (M), a primer signal detection region (h) and a target sequence binding region, the extension block region (M) being a freely designed sequence that is neither identical nor complementary to any part of the sequence of the probe (P) or any target sequence, and if there is another type of reverse primer, the 5' end of the other type of reverse primer (R) does not comprise the extension block region (M).

[0060] In some embodiments, if there is another type of reverse primer, the other type of reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 3' end of the target sequence binding region. In some embodiments, if there is another type of reverse primer, the other type of reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region in the order from the 5' end to the 3' end.

[0061] According to a preferred embodiment of the present invention, in the primers, at least one type of forward primer (F) further comprises a probe anchor region (A), the probe anchor region (A) in the forward primer (F) is located at the 5' end of the target sequence binding region, wherein the probe anchor region (A) is a sequence that does not pair with and bind to any target sequence, and the sequences of the target sequence binding regions of different forward primers (F) are different, and more preferably, there is an interval of 0-20 bases between the probe anchor region (A) and the target sequence binding region in the forward primer (F), wherein the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by these, The probe (P) is a sequence that does not pair with any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H), the primer anchor region (A') being a sequence complementary to the probe anchor region (A) of the forward primer (F), and more preferably, there is an interval of 0-20 bases between the primer anchor region (A') and the probe signal detection region (H) in the probe (P). Here, the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by these.

[0062] According to a preferred embodiment of the present invention, in the primers, at least one type of forward primer (F) comprises a probe anchor region (A) and a target sequence binding region in this order from the 5' end to the 3' end, wherein the probe anchor region (A) is a sequence that does not bind to any target sequence, and the sequences of the target sequence binding regions of different forward primers (F) are different, and preferably, there is an interval of 0-20 bases between the probe anchor region (A) and the target sequence binding region in the forward primer (F), wherein the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by these, The probe (P) is a sequence that does not bind to any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H), the primer anchor region (A') is a sequence that is reverse complementary to the probe anchor region (A) of the forward primer (F), and preferably, there is an interval of 0 to 20 bases between the primer anchor region (A') and the probe signal detection region (H) in the probe (P). Here, the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them.

[0063] According to a preferred embodiment of the present invention, in the primer, at least one of the forward primers (F) includes a first forward primer and a second forward primer, the first forward primer includes a forward amplification product capture region (B) and a target sequence binding region from the 5' end to the 3' end in this order, and the second forward primer includes a forward amplification product capture region (B), the forward amplification product capture region (B) does not bind to the target sequence or any fragment thereof, and more preferably, there is an interval of 0-20 bases between the forward amplification product capture region (B) and the target sequence binding region in the first forward primer. Here, the number of bases of the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them.

[0064] According to a preferred embodiment of the present invention, the second forward primer further comprises a probe anchor region (A), and preferably there is a 0-20 base interval between the probe anchor region (A) and the forward amplification product capture region (B) in the second forward primer.

[0065] According to a preferred embodiment of the present invention, in the primer, at least one of the reverse primers (R) includes a first reverse primer and a second reverse primer, the first reverse primer includes, from the 5' end to the 3' end, a reverse amplification product capture region (C) and a target sequence binding region, and the second reverse primer includes, from the 5' end to the 3' end, a primer signal detection region (h) and a reverse amplification product capture region (C), the reverse amplification product capture region (C) does not pair with and bind to a target sequence or any fragment thereof, Preferably, there is a 0-20 base interval between the reverse amplification product capture region (C) and the target sequence binding region in the first reverse primer, and / or there is a 0-20 base interval between the primer signal detection region (h) and the reverse amplification product capture region (C) in the second reverse primer, where the number of bases in the interval may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them.

[0066] In some embodiments of the present invention, the method for detecting two or more target nucleic acids is a digital PCR detection method, and in some embodiments of the present invention, before performing a first PCR amplification on the reaction system, the method further includes distributing the reaction system into 500 or more reaction cells, such that each reaction cell contains one target nucleic acid of a test sample or does not contain any target nucleic acid of a test sample, and preferably, the signal collection is collecting a fluorescent signal by a camera, and the signal channel collection is collecting a fluorescent signal by a camera in a fluorescent signal channel.

[0067] In the above-mentioned method for detecting a target nucleic acid, the primer probe composition may be a conventional primer probe composition in the present field, for example, a primer probe composition containing a molecular beacon, or a primer probe composition for detecting a nucleic acid sequence provided in the third aspect described below.

[0068] Therefore, in a third aspect, the present invention provides a primer probe composition for detecting a nucleic acid sequence, comprising a first probe and a first primer mixture. The first primer mixture contains at least two types of primer sets, and each of the different types of primer sets specifically binds to a different type of target nucleic acid. The primer sets in the first primer mixture generate pre-products after specifically binding to their corresponding target nucleic acids, the pre-products include single-stranded pre-products that specifically bind to a first probe, the single-stranded pre-products form double-stranded products after specifically binding to the first probe and extending by ≧0 bases, and the formation of the double-stranded products causes a detectable signal change. Different types of primer sets in the first primer mixture produce different single-stranded pre-products with their corresponding target nucleic acids, and when the annealing temperature of the first single-stranded pre-product and the first probe is higher than the annealing temperature of the second single-stranded pre-product and the first probe, the melting temperature of the double-stranded product produced by the first single-stranded pre-product and the first probe is lower than the melting temperature of the double-stranded product produced by the second single-stranded pre-product and the first probe.

[0069] The first probe is one type of probe that has the same modified detection label and may have the same or different base sequence. That is, the number of types of probes is classified according to the number of types of detection labels. In some embodiments, the signal generated by one type of probe can be collected in one detection channel (or fluorescence channel), and the signals generated by two types of probes need to be collected in two different detection channels. In some embodiments, the detection label being the same does not mean that the modified detection groups are completely the same, for example, if the detection group includes a fluorescent group and a quenching group and the fluorescent groups are the same, the signal generated by the probe can be collected in one detection channel (or fluorescence channel).

[0070] "At least two types of primer sets" means that there are at least two types of primer sets, and each primer set has a different type of target nucleic acid, that is, each primer set can specifically bind to its corresponding target nucleic acid. In some embodiments, if one type of primer set can specifically bind to another type of non-differentiated target nucleic acid, this type of non-differentiated target nucleic acid can also be considered as one type of target nucleic acid.

[0071] In some embodiments, the primer set is a double primer, i.e., includes a pair of upstream and downstream primers for the same target nucleic acid. In some embodiments, the primer set is a single primer, i.e., includes only one type of primer for a target nucleic acid, which can specifically bind to its corresponding target nucleic acid and cannot specifically bind to other types of target nucleic acids.

[0072] In some embodiments, different types of primer sets may have a common primer, for example, two types of primer sets each contain one similar primer, which means that the primers have exactly the same sequence.

[0073] In general, the annealing temperature of an oligonucleotide is usually about 5°C lower than the melting temperature (Tm) of the oligonucleotide, but can be determined to be about 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0°C or lower than the Tm of the oligonucleotide, or any range therebetween (including the end values). The annealing temperature and melting temperature of an oligonucleotide are related to factors such as the length and composition of the oligonucleotide. Those skilled in the art can adjust the annealing temperature and melting temperature of an oligonucleotide according to actual needs, for example, a higher melting temperature can be obtained by increasing the GC content of the oligonucleotide or increasing the length of the oligonucleotide.

[0074] In some embodiments, the lower the annealing temperature required for a single-stranded pre-product generated by a primer set and its target nucleic acid to specifically bind to a probe, the higher the melting temperature of the double-stranded product formed by the single-stranded pre-product specifically binding to the probe and extending by ≧0 bases. For example, the length and / or sequence of the primer set is adjusted to obtain different single-stranded pre-products obtained by specifically binding the primer set to the target nucleic acid. The different binding positions of the different single-stranded pre-products and the same probe make it possible to realize that the annealing temperature between the first single-stranded pre-product and the probe (abbreviated as "first annealing temperature" herein) is higher than the annealing temperature between the second single-stranded pre-product and the probe (abbreviated as "second annealing temperature" herein), i.e., the first annealing temperature is higher than the second annealing temperature. Furthermore, since the binding positions of different single-stranded pre-products and the same probe are different, it is also possible to adjust the length of extension of different single-stranded pre-products by binding to the probe, and to realize that the melting temperature of the double-stranded product generated by the first single-stranded pre-product and the probe (abbreviated here as the "first melting temperature") is lower than the melting temperature of the double-stranded product generated by the second single-stranded pre-product and the probe (abbreviated here as the "second melting temperature"), i.e., the first melting temperature is lower than the second melting temperature.

[0075] In the present invention, the terms "higher" and "lower" are used to describe that there is a difference between two temperatures, and due to the existence of the difference, different types of single-stranded pre-products can achieve different states of single-stranded or double-stranded when collecting signals at a certain temperature, for example, at a certain signal temperature, the first single-stranded pre-product exhibits a single-stranded state, and the second single-stranded pre-product binds to the probe and exhibits a double-stranded state. In some embodiments, "higher" is used to describe that the difference between the two annealing temperatures is 4°C or more, for example, the difference is 4-40°C, and also, for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, or 4-5°C, etc. In some embodiments, "lower than" is used to describe a difference between two melting temperatures of 6°C or more, such as a difference of 6-46°C, such as a difference of 6-40°C, 6-35°C, 6-30°C, 6-25°C, 6-20°C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C or 6-7°C.

[0076] By using the above primer probe composition and setting the difference between high and low annealing temperature and melting temperature, when collecting signals at a certain temperature, at most one type of single-stranded pre-product is in a double-stranded state with the probe, so that at most one type of target nucleic acid signal is generated (the above "at most" means that if the target nucleic acid is not present in the test sample, no signal is generated, and if it is present, only the signal of the target nucleic acid is generated, and no other target nucleic acid signal is generated at the same time). Therefore, it is realized that signals collected at the same temperature do not overlap.

[0077] In some embodiments, in the primer-probe composition, there is one type of probe (the first probe) and two types of primer sets. The two types of primer sets are the first primer set and the second primer set respectively. After the first primer set and the second primer set specifically bind to the first target nucleic acid and the second target nucleic acid respectively, they generate the first single-stranded pre-product and the second single-stranded pre-product respectively. The annealing temperature of the first single-stranded pre-product and the probe is the first annealing temperature, and the annealing temperature of the second single-stranded pre-product and the probe is the second annealing temperature. The double-stranded product generated by the first single-stranded pre-product and the probe is the first double-stranded product, and its melting temperature is the first melting temperature. The double-stranded product generated by the second single-stranded pre-product and the probe is the second double-stranded product, and its melting temperature is the second melting temperature. After performing a PCR cycle at an annealing temperature of T1 (the first annealing temperature ≥ T1 > the second annealing temperature), only the first double-stranded product is generated, and the first signal collection is performed at the temperature of t1 (t1 < the first melting temperature). At this time, only the first double-stranded product is in the double-stranded state, and the generated signal is the signal of the first target nucleic acid. Then, after performing a PCR cycle at an annealing temperature of T2 (the second annealing temperature ≥ T2), the second double-stranded product is generated, and the reaction system further contains the first double-stranded product. Then, the second signal collection is performed at the temperature of t2 (the first melting temperature < t2 < the second melting temperature). At this time, only the second double-stranded product is in the double-stranded state, and the generated signal is the signal of the second target nucleic acid (because t2 > the first melting temperature, the first double-stranded product is in the denatured state and no signal is generated).

[0078] Similarly, when three or more kinds of primer sets are included, when a certain temperature is used as the PCR annealing temperature to carry out PCR cycle amplification, all single-stranded pre-products with annealing temperatures higher than the temperature can bind with the probe to form double-stranded products, and single-stranded pre-products with annealing temperatures lower than the temperature remain in single-stranded state.When a certain temperature is used to collect signals, only one kind of single-stranded pre-product is double-stranded with the probe to generate a detectable target nucleic acid signal.Some of the other kinds of single-stranded pre-products have low annealing temperatures, so they cannot anneal with the probe to form double-stranded products in the PCR cycle amplification stage, and some of them have high annealing temperatures, so they can anneal with the probe to form double-stranded products in the PCR cycle amplification stage, but the melting temperatures of these double-stranded products are lower than the temperature of signal collection, so the double-stranded products generated are also in single-stranded state at the signal collection temperature.

[0079] In the present invention, expressions such as "first," "second," "third," "fourth," and "fifth" are used for explanatory purposes only and are used to distinguish limited objects, and do not limit the order or hierarchy in any way.

[0080] In some embodiments, the primer probe composition for detecting a target nucleic acid includes a first primer set for a first target nucleic acid, the first primer set generates a first pre-product after specifically binding to the first target nucleic acid, the first pre-product includes a first single-stranded pre-product that specifically binds to a probe, the first single-stranded pre-product forms a first double-stranded product after specifically binding to the probe and extending by 0 bases, and the formation of the first double-stranded product causes a detectable signal change. The annealing temperature of the first single-stranded pre-product and the probe is higher than the annealing temperature of the same probe with other single-stranded pre-products, and the melting temperature of the first double-stranded product generated by the first single-stranded pre-product and the probe is lower than the melting temperature of the double-stranded product generated by the same probe with other single-stranded pre-products.

[0081] In this embodiment, a primer set for a certain target nucleic acid (herein referred to as "first primer set") is not extended after the generated first single-stranded pre-product specifically binds to the probe. Those skilled in the art can achieve the above-mentioned effect of "specifically binding but not extending" by designing the primer sequence in the primer set and / or the sequence of the probe according to actual needs, for example, by designing the generated first single-stranded pre-product to bind to the 5' end of the probe, or by designing the generated first single-stranded pre-product to include a region at the 3' end that does not complementarily pair with the probe.

[0082] The above "first" is used only for the purpose of explanation and distinction, and is used to distinguish limited objects, and is not used to limit the order or hierarchy, and is not used to limit the number of types of target nucleic acids, primer sets, pre-products, single-stranded pre-products, or double-stranded products. Here, it is used only to explain that in the above-mentioned primer probe composition, a single-stranded pre-product generated by one type of primer set may not be extended after specifically binding to a probe, and the annealing temperature between the single-stranded pre-product and the probe is the highest (higher than the annealing temperature between any other type of single-stranded pre-product and the same probe), and the melting temperature of the formed double-stranded product is the lowest (lower than the melting temperature of any other type of double-stranded product).

[0083] In some embodiments of the present invention, the primer probe composition for detecting a target nucleic acid described above further comprises a second probe and a second primer mixture, the second probe having a base sequence different from that of the first probe and also having a modified detection label different from that of the first probe, the second primer mixture comprising at least one type of primer set, and the different types of primer sets each specifically bind to a different type of target nucleic acid.

[0084] In this embodiment, the primer probe composition includes another type of probe (second probe). The number of types of probes is classified according to the number of types of their detection labels. Different types of probes have different detection labels, so it is necessary to perform signal collection in different detection channels. In some embodiments, different detection labels do not mean that the modified detection groups are completely different, for example, as long as the detection group includes a fluorescent group and a quenching group and the fluorescent groups are different (the quenching groups may be the same or different), it is also possible to realize signal collection of the signals generated by the two types of probes in two detection channels (or fluorescent channels). In this embodiment, by increasing the number of types of probes, it is possible to realize signal detection in different channels for different target nucleic acids, and to realize detection of more multiplexed target nucleic acids.

[0085] In this embodiment, the terms "first probe" and "second probe" are used, but they are not used as a limiting role, nor do they limit the present embodiment to only two types of probes. Here, the "first probe" and the "second probe" are used to distinguish that they belong to different types of probes, and the detection labels modified by the two types of probes are different and the base sequences are also different. In some embodiments, in order to detect more types of target nucleic acids, the primer-probe composition for detecting target nucleic acids further includes a third probe and a third primer mixture, a fourth probe and a fourth primer mixture, a fifth probe and a fifth primer mixture, a sixth probe and a sixth primer mixture, or more probes and primer mixtures. The detection labels modified by different probes are different and the base sequences are also different, and the primer sets in different primer mixtures are also different. That is, in order to realize the detection of more multiple target nucleic acids, those skilled in the art can design more types of primer sets corresponding to target nucleic acids and more types of probes corresponding to primer sets as needed.

[0086] In some embodiments of the present invention, the above-mentioned primer probe composition for detecting a target nucleic acid generates a pre-product after the primer set in the second primer mixture specifically binds to the corresponding target nucleic acid, the pre-product includes a single-stranded pre-product that specifically binds to the second probe, the single-stranded pre-product specifically binds to the second probe and forms a double-stranded product after being extended by ≧0 bases, and the formation of the double-stranded product causes a detectable signal change.

[0087] Preferably, different types of primer sets in the second primer mixture have different single-stranded pre-products generated by their corresponding target nucleic acids, and when the annealing temperature of the third single-stranded pre-product and the second probe is higher than the annealing temperature of the fourth single-stranded pre-product and the second probe, the melting temperature of the double-stranded product generated by the third single-stranded pre-product and the second probe is lower than the melting temperature of the double-stranded product generated by the fourth single-stranded pre-product and the second probe.

[0088] By setting the high-low relationship between the annealing temperature and the melting temperature, it is possible to realize that the signal collected at a certain detection temperature in a certain detection channel is the signal of at most one type of target nucleic acid (the above-mentioned "at most" means that if the target nucleic acid is not present in the test sample, no signal is generated, and if it is present, only the signal of that type of target nucleic acid is present, and signals of other target nucleic acids do not appear simultaneously).

[0089] In the present invention, the terms "third", "fourth", etc. are used for explanatory purposes only, and are used to distinguish limited objects, and do not limit the order or hierarchy. In this embodiment, "third" is used to distinguish from "fourth", that is, the "third single-stranded pre-product" and the "fourth single-stranded pre-product" belong to different types and correspond to different types of target nucleic acid.

[0090] In the present invention, the terms "third", "fourth", etc. are used only for the purpose of explanation, and are used to distinguish limited objects, and are not used to limit the number of types of single-stranded pre-products. For example, in this embodiment, "fourth single-stranded pre-product" appears, but it does not limit that there are only four types of target nucleic acids in this embodiment. In some embodiments, four or more target nucleic acids are further included to generate four or more single-stranded pre-products. In some embodiments, the second primer mixture includes three or more primer sets, and the annealing temperature and melting temperature of the single-stranded pre-products generated by these primer sets and their corresponding target nucleic acids are similar to those of the third single-stranded pre-product and the fourth single-stranded pre-product, that is, the lower the annealing temperature required for the single-stranded pre-product generated by a certain type of primer set and its target nucleic acid to specifically bind to the probe, the higher the melting temperature of the double-stranded product formed by the single-stranded pre-product specifically binding to the probe and extending by ≧0 bases. When a certain temperature is used as the PCR annealing temperature for PCR cycle amplification, all single-stranded pre-products with annealing temperatures higher than this temperature can combine with the second probe to form double-stranded products, and single-stranded pre-products with annealing temperatures lower than this temperature remain in single-stranded state; when a certain temperature is used to collect signals, only one type of single-stranded pre-product can be double-stranded with the second probe to generate detectable target nucleic acid signals. Other types of single-stranded pre-products, some of which have low annealing temperatures, cannot anneal with the second probe to form double-stranded products in the PCR cycle amplification stage, and the other types of single-stranded pre-products, have high annealing temperatures, and can anneal with the second probe to form double-stranded products in the PCR cycle amplification stage, but the melting temperatures of these double-stranded products are lower than the temperature of signal collection, so the double-stranded products generated are also single-stranded at the signal collection temperature.

[0091] In some embodiments of the present invention, in the above-mentioned primer probe composition for detecting a target nucleic acid, the melting temperature of the double-stranded product generated by the first single-stranded pre-product and the first probe is equal to or close to the melting temperature of the double-stranded product generated by the third single-stranded pre-product and the second probe, and preferably, the annealing temperature of the first single-stranded pre-product and the first probe is equal to or close to the annealing temperature of the third single-stranded pre-product and the second probe.

[0092] In this embodiment, the primer probe composition contains two or more kinds of probes. The number of types of probes is classified according to the number of types of detection labels. Different types of probes have different detection labels, so they need to be collected by different detection channels.

[0093] In the present invention, the terms "equal" and "close" are used to explain that there is no difference or the difference is small between the two temperatures. Also, because the difference is small or there is no difference, different types of single-stranded pre-products realize the same single-stranded or double-stranded state when signals are collected at a certain temperature. For example, at a certain temperature, the first single-stranded pre-product and the third single-stranded pre-product are both in a single-stranded state, or the first single-stranded pre-product and the third single-stranded pre-product are both in a double-stranded state. Note that when they are collected at a certain temperature, they can all maintain a double-stranded state, but the types of probes bound to them when they are in a double-stranded state are different. In some embodiments, "equal" and "close" are used to explain that the difference between the two annealing temperatures / two melting temperatures is within 4°C, for example, the difference is 0-4°C, preferably the difference is 0-2°C, and more preferably the difference is 0-1°C.

[0094] In this embodiment, the terms "first", "second", "third", etc. are used only for the purpose of explanation, and are used to distinguish limited objects, and do not limit the order or the order of the single-stranded pre-products or the number of types of probes. For example, in this embodiment, a "third single-stranded pre-product" appears, but this does not limit the fact that there are only three types of target nucleic acids in this embodiment, and only three types of single-stranded pre-products. In this embodiment, "third" is used simply to distinguish from "first", that is, the "first single-stranded pre-product" and the "third single-stranded pre-product" belong to different types and the corresponding types of target nucleic acids are different. In addition, for example, in this embodiment, "first probe" and "second probe" appear, but this does not limit the fact that there are only two types of probes in this embodiment, and here, "first probe" and "second probe" are simply used to distinguish that they belong to different types of probes, and the detection groups modified by the two types of probes are different, and the base sequences are also different. This embodiment also includes the case of three types or more than three types of probes. To achieve detection of more multiplexed target nucleic acids, one skilled in the art can design more types of probes as needed.

[0095] By setting the melting temperatures to be the same or close to each other, when signal collection is performed at a certain temperature, two or more types of single-stranded pre-products and probes can be in a double-stranded state. However, since the types of probes bound to different single-stranded pre-products that exhibit a double-stranded state at this temperature are different, the same temperature is adopted, but signals are collected in different detection channels, thereby making it possible to distinguish target nucleic acids corresponding to different single-stranded pre-products. In this way, by increasing the number of detection channels (fluorescence channels), it becomes possible to detect more multiplexed nucleic acids. In addition, the number of times the signal collection temperature is changed can be reduced, that is, different targets can be detected at a certain signal collection temperature simply by changing the detection channel, and the number of PCR cycles can also be reduced, that is, after a certain PCR cycle, the number of types of double-stranded products formed per type of probe increases by at most one type compared to the previous PCR amplification (if there are two types of probes, two types of double-stranded products increase).

[0096] However, it is not the case that two or more types of single-stranded pre-products and probes can be in a double-stranded state when collecting signals at a certain temperature only when the melting temperatures are set to be the same or close to each other (different types of probes are bound to different single-stranded pre-products that exhibit a double-stranded state). When the melting temperatures are not the same or close to each other (for example, the difference between the two is 6°C or more), the above effect can be achieved by adjusting the temperature of signal collection to be lower than the melting temperatures of different double-stranded products generated by different single-stranded pre-products and different types of probes. For example, if the melting temperature Tm1 of the double-stranded product generated by the first single-stranded pre-product and the first probe is not equal to or close to the melting temperature Tm2 of the double-stranded product generated by the third single-stranded pre-product and the second probe, and the temperature of signal collection is adjusted to be lower than Tm1 and Tm2, ​​the two double-stranded products will still be in a double-stranded state at the temperature of signal collection.

[0097] In some embodiments of the present invention, in the above-mentioned primer probe composition for detecting a target nucleic acid, the first probe or the second probe has a sequence that does not specifically bind to any target nucleic acid and includes a probe signal detection region (H), and the sequences of the probe signal detection regions (H) of different probes are different from each other.

[0098] The primer set comprises a first primer including a target sequence binding region 1, and a second primer including a primer signal detection region (h) and a target sequence binding region 2, with the primer signal detection region (h) located at the 5' end of the target sequence binding region 2, wherein the primer signal detection region (h) does not specifically bind to any target nucleic acid and has a sequence that is the same in part or in whole as the probe signal detection region (H) of the corresponding probe, and the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other.

[0099] In some embodiments, target sequence binding region 1 and target sequence binding region 2 each specifically bind to a different location on the target nucleic acid.

[0100] In some embodiments, the primer signal detection region (h) having a sequence that is partially or completely identical to the probe signal detection region (H) of the probe means that the reverse complementary sequence (h') of the primer signal detection region (h) can specifically bind to a portion or all of the sequence of the probe signal detection region (H) of the probe.

[0101] In some embodiments, the above-mentioned first primer and second primer specifically bind to the target nucleic acid, and then generate a pre-product comprising one single-stranded pre-product having a reverse complementary sequence (h') of the primer signal detection region (h), and the reverse complementary sequence (h') of the single-stranded pre-product specifically binds to the probe signal detection region (H) of the probe, and then extends by ≧0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change.

[0102] In some embodiments, the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other, i.e., the bases and / or lengths of the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different, so that the sequences of the reverse complementary sequences (h') of the different single-stranded pre-products are different, and the different single-stranded pre-products form different double-stranded products after specifically binding to the probe and extending 0 or >0 bases, and the melting temperatures of the different double-stranded products can be separated from each other. Or, the different single-stranded pre-products form different double-stranded products after specifically binding to different types of probes and extending ≧0 bases, and the melting temperatures of the different double-stranded products cannot be separated from each other, but because the types of probes for the different double-stranded products are different, they can be distinguished by different detection channels. That is, different types of target nucleic acids can be distinguished by melting temperatures and / or detection channels to realize multiplex detection.

[0103] In some embodiments, there is a 0-20 base interval between the primer signal detection region (h) of the second primer and the target sequence binding region 2, where the number of bases in the interval can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range formed therewith.

[0104] In some embodiments of the present invention, in the above-mentioned primer probe composition for detecting a target nucleic acid, the first probe or the second probe further contains a primer anchor region (A'), and the first primer of at least one primer set of the first primer mixture or the second primer mixture further contains a probe anchor region (A), which is located at the 5' end of the target sequence binding region 1, and which does not specifically bind to any target nucleic acid but specifically binds to the primer anchor region (A').

[0105] The first and second primers in the above primer set specifically bind to the target nucleic acid and then generate a pre-product, which includes one single-stranded pre-product having a probe anchor region (A) and a reverse complementary sequence (h') of the primer signal detection region (h), and the probe anchor region (A) and the reverse complementary sequence (h') of the single-stranded pre-product specifically bind to the primer anchor region (A') and the probe signal detection region (H) of the probe, respectively, and form a double-stranded product after being extended by ≧0 bases, and the formation of the double-stranded product causes the probe to produce a detectable signal change.

[0106] By adjusting the sequence composition and / or length of the probe anchor region (A) of the first primer in the different primer sets and / or the primer signal detection region (h) of the second primer in the different primer sets, the single-stranded pre-products generated by the different primer sets and their target nucleic acids are different, the annealing temperatures generated by the specific binding of the different single-stranded pre-products to the probe are different, and the melting temperatures of the double-stranded products formed after the specific binding and extension by ≧0 bases are also different; the lower the annealing temperature required for the specific binding of the single-stranded pre-product generated by a certain primer set and its target nucleic acid to the probe, the higher the melting temperature of the double-stranded product formed after the single-stranded pre-product is specifically bound to the probe and extended by ≧0 bases.

[0107] In some embodiments, the sequences of the target sequence binding region 1 of the first primer in different primer sets are different. The sequences of the probe anchor region (A) of the first primer in different primer sets may be the same or different. Preferably, there is a gap of 0-20 bases between the probe anchor region (A) of the first primer and the target sequence binding region 1. Here, the number of bases of the gap may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range constituted by them.

[0108] In some embodiments of the present invention, the above-mentioned primer probe composition for detecting a target nucleic acid, in the first primer mixture or the second primer mixture, the second primer in at most one type of primer set further contains an extension block region (M) located at the 5' end of the primer signal detection region (h), and neither the extension block region (M) nor its complementary sequence specifically binds to any probe or any target nucleic acid.

[0109] The first primer and the second primer including the extension block region (M) each generate a pre-product after specifically binding to the target nucleic acid, and the pre-product includes one single-stranded pre-product having a reverse complementary sequence (h') of the primer signal detection region (h), and the reverse complementary sequence (h') of the single-stranded pre-product specifically binds to the probe and extends by 0 bases to form a double-stranded product (due to the presence of the extension block region, the single-stranded pre-product is not extended after specifically binding to the probe), causing the probe to produce a detectable signal change.

[0110] In some embodiments of the present invention, after the primer specifically binds to the target nucleic acid, the generated single-stranded pre-product is not extended after specifically binding to the probe (i.e., extended by 0 bases). Those skilled in the art can achieve the above effect of "specifically binding but not extending" by designing the primer sequence in the primer set and / or the sequence of the probe according to actual needs. For example, the generated first single-stranded pre-product is designed to bind to the 5' end of the probe, or the generated first single-stranded pre-product is designed to include a region at the 3' end that is not complementary to the probe.

[0111] The terms "first primer mixture", "second primer mixture", etc. are used for explanatory purposes only and to distinguish limited objects. Both refer to a set of primer mixtures that specifically bind to a probe, and the primer mixture contains multiple types of primer sets, and the reverse complementary sequences (h') of the primer signal detection regions (h) contained in the second primers in these primer sets can all specifically bind to the same type of probe. However, the types of probes to which the primer sets specifically bind are different between the "first primer mixture" and the "second primer mixture".

[0112] In some embodiments, in a mixture of primers that specifically bind to the same type of probe, the second primer in at most one type of primer set contains an extension block region (M), and the single-stranded pre-product generated after this type of primer set specifically binds to the target nucleic acid has the highest annealing temperature required to specifically bind to the probe, and the double-stranded product generated after specifically binding to the probe without extension has the lowest melting temperature.

[0113] In the present invention, the terms "first primer" and "second primer" are used for explanatory purposes only, and are used to distinguish between limited objects, and do not limit the order or hierarchical order. For example, in the above primer probe composition, the structures of the first primer and the second primer of the primer set are interchangeable, for example, the first primer comprises a primer signal detection region (h) and a target sequence binding region, and the second primer comprises a target sequence binding region, and for example, the first primer comprises a primer signal detection region (h) and a target sequence binding region, and the second primer comprises a probe anchor region (A) and a target sequence binding region. In some embodiments, the "first primer" is also called a "forward primer" and the "second primer" is also called a "reverse primer".

[0114] In the present invention, the probe is a freely designed sequence that does not pair with any target nucleic acid, and the probe is modified with a detection label. Preferably, the detection label includes a first detection group and a second detection group, and the first detection group and the second detection group cause a change in signal due to a change in distance. Preferably, the first detection group and the second detection group are spaced apart by 3-250 angstroms, preferably by 3-201 angstroms, more preferably by 3-140 angstroms, and / or the first detection group is a fluorescent reporter group, and the second detection group is a quenching group or a modification group that can cause a signal change by fluorescence resonance energy transfer with another first detection group.

[0115] In the present invention, depending on the positions of the first and second detection groups in the probe, the reverse complementary sequence (h') of the single-stranded pre-product and the probe (P) specifically bind to each other to form a double-stranded product after elongation by ≧0 bases, and if the positions of the first and second detection groups change due to the formation of the double-stranded product, a detectable signal change can be caused in the probe. The positions of the first and second detection groups are interchangeable.

[0116] In some embodiments, when the target nucleic acid to be detected is not present, there is no specific binding between the single-stranded pre-product and the probe, and the probe is in a single-stranded state or in another secondary structure, in which case the distance between the first detection group and the second detection group is close, and the efficiency of fluorescence resonance energy transfer is high. When the first primer contains a probe anchor region (A), even if the primer anchor region (A') of the probe and the probe anchor region (A) in the first primer are complementary, the other part of the probe is in a single-stranded state or in another secondary structure, in which case the distance between the first detection group and the second detection group is still close, and the efficiency of fluorescence resonance energy transfer is high. When the target nucleic acid is present, the first primer and the second primer each specifically bind to the target sequence and extend to generate double-stranded amplification products, of which one single-stranded amplification product specifically binds to the probe to form a double-stranded product, in which case the distance between the first detection group and the second detection group is increased, the efficiency of fluorescence resonance energy transfer is reduced, and the fluorescent signal is changed and detected by the instrument.

[0117] Therefore, in a fourth aspect, the present invention provides a PCR detection kit comprising the primer-probe composition for detecting a target nucleic acid of the third aspect. Those skilled in the art know that in order to realize PCR detection, the kit further comprises an amplification reagent for amplification, such as DNA polymerase and dNTPs. When the nucleic acid template is RNA, the amplification reagent further comprises reverse transcriptase. The amplification reagent further comprises some reagents for facilitating the PCR reaction, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, etc. In some preferred embodiments, the PCR detection kit according to the present invention is a digital PCR detection kit comprising the primer-probe composition for detecting a target nucleic acid of the third aspect.

[0118] Therefore, in a fifth aspect, the present invention provides a reaction liquid storage section for storing a plurality of micro-fluids including a sample and a reagent; A temperature control unit for controlling the temperature of the micro-liquid in the reaction liquid storage unit; A signal detection unit for detecting a signal generated by the micro liquid in the reaction liquid storage unit; The temperature control unit is controlled to control the temperature of the reaction liquid storage unit in a first temperature control mode, and a valid signal is generated only in a first portion of the micro-liquids among the plurality of micro-liquids, and the signal detection unit is controlled to perform signal detection on the reaction liquid storage unit to obtain a first signal; and a control unit that controls the temperature adjustment unit to adjust the temperature of the reaction liquid storage unit in a second temperature adjustment mode, generates a valid signal only in a second portion of micro-liquids that are different from the micro-liquids in the first portion among a plurality of micro-liquids stored in the same reaction liquid storage unit, and controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a second signal.

[0119] The microfluidic device is a mixture containing a sample and a reagent.

[0120] The first and second temperature regulation modes are different. In some embodiments, different means that the minimum temperatures used in the two temperature regulation modes are different. In some embodiments, the minimum temperature used in the first temperature regulation mode is higher than the minimum temperature used in the second temperature regulation mode.

[0121] In one embodiment, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to a temperature t2, and controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a first signal; The control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to temperature t3, and controls the signal detection unit to perform signal detection for the reaction liquid storage unit to obtain a second signal. However, t2 <t3である。

[0122] In some embodiments, the difference in temperature between t2 and t3 is 6°C or more, such as, for example, the difference is 6-46°C, including, for example, the difference is 6-40°C, 6-35°C, 6-30°C, 6-25°C, 6-20°C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C, or 6-7°C.

[0123] In one embodiment thereof, the reagent comprises a first reagent and a second reagent; When the temperature of the micro-fluid in the reaction liquid storage section is t2, the first reagent in the micro-fluid in the first portion combines with the sample to generate a signal; When the temperature of the micro-fluid in the reaction liquid storage section is t3, the second reagent in the micro-fluid in the second section combines with the sample to generate a signal.

[0124] In another embodiment, the first temperature regulation mode includes an incubation phase and a signal collection phase; In the incubation stage, the temperature control unit periodically changes the temperature of the micro-liquid in the reaction liquid storage unit so that the reaction liquid storage unit is repeatedly lowered from a high temperature to a low temperature T2, or the temperature control unit maintains the reaction liquid storage unit at a constant temperature T2; In the signal collecting step, the signal detection unit performs signal detection on the reaction liquid storage unit to obtain a first signal.

[0125] In one embodiment thereof, the second temperature regulation mode includes an incubation phase and a signal collection phase; In the incubation stage, the temperature of the micro-liquid in the reaction liquid storage unit is periodically changed so that the reaction liquid storage unit is repeatedly lowered from a high temperature to a low temperature T3, or the temperature control unit maintains the reaction liquid storage unit at a constant temperature T3; In a signal collecting step, the signal detection unit performs signal detection on the reaction liquid storage unit to obtain a second signal; However, T2>T3 and t2>T3.

[0126] In some embodiments, the difference in temperature between T2 and T3 is 4°C or more, for example, the difference is 4-40°C, including, for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, or 4-5°C.

[0127] In some embodiments, the difference in temperature between t2 and T3 is 5°C or more, for example, the difference is 5-50°C, including, for example, the difference is 5-40°C, 5-30°C, 5-20°C, 5-18°C, 5-15°C, 5-12°C, 5-10°C, 5-8°C, 5-7°C, or 5-6°C.

[0128] In one embodiment, when the temperature of the micro-fluid in the reaction liquid receiving portion is T2, the first reagent in the micro-fluid in the first portion combines with the sample to generate a signal; When the temperature of the micro-fluid in the reaction liquid storage section is T3, the first reagent in the micro-fluid in the first section binds to the sample to generate a signal, and the second reagent in the micro-fluid in the second section binds to the sample to generate a signal.

[0129] In one embodiment thereof, before the control unit controls the temperature control unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit in the first temperature control mode and the second temperature control mode, The control unit controls the temperature adjustment unit to periodically change the temperature of the micro-liquid in the reaction liquid storage unit, thereby repeatedly lowering the temperature of the reaction liquid storage unit from a high temperature to a low temperature T1, and generating a valid signal only in the micro-liquid in a fourth portion of the multiple micro-liquids, the micro-liquid in the fourth portion being different from both the micro-liquid in the first portion and the micro-liquid in the second portion, with the proviso that T1>T2.

[0130] In some embodiments, the difference in temperature between T1 and T2 is 4°C or more, for example, the difference is 4-40°C, such as, for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, or 4-5°C.

[0131] In one embodiment thereof, the reagent further comprises a fourth reagent, When the temperature of the micro-fluid in the reaction liquid storage section is T1, the fourth reagent in the micro-fluid in the fourth portion combines with the sample to generate a signal; When the temperature of the micro-fluid in the reaction liquid storage section is T2, the fourth reagent in the micro-fluid in the fourth portion combines with the sample to generate a signal, and the first reagent in the micro-fluid in the first portion combines with the sample to generate a signal; When the temperature of the micro-fluid in the reaction liquid storage section is T3, the fourth reagent in the micro-fluid in the fourth section binds to the sample to generate a signal, the first reagent in the micro-fluid in the first section binds to the sample to generate a signal, and the second reagent in the micro-fluid in the second section binds to the sample to generate a signal.

[0132] In one embodiment, the signal detection unit is controlled to perform signal detection for the reaction liquid storage unit to obtain a fourth signal.

[0133]

[0134] In one embodiment, in the process in which the signal detection unit detects the signal in the reaction liquid storage unit and obtains a fourth signal, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to t1 or maintain the temperature at T1; When the temperature of the micro-fluid in the reaction liquid storage section is at T1 or t1, the fourth reagent in the micro-fluid in the fourth portion remains bound to the sample and continues to generate a signal.

[0135] In one embodiment thereof, at most one unit of sample is dispensed into the microfluidic device in an initial state.

[0136] In one embodiment thereof, the signal is a fluorescent signal and the signal detector is a camera.

[0137] In one embodiment, the control unit controls the signal detection unit to perform signal detection for the reaction liquid container at a position where the micro-liquids are spread out in the reaction liquid container.

[0138] In one embodiment, the control unit controls the temperature adjustment unit to adjust the temperature of the reaction liquid storage unit at a position where the micro-liquid particles are spread in the reaction liquid storage unit.

[0139] Therefore, in a sixth aspect, the present invention provides a method for detecting a first signal by controlling the temperature of a plurality of micro-fluids in a first temperature control mode, and detecting the first signal by detecting the first signal from the plurality of micro-fluids; The present invention provides a detection method for detecting a plurality of micro-fluids containing a sample and a reagent, the method comprising the steps of: adjusting the temperature of the micro-fluid in a second temperature adjustment mode, thereby generating a valid signal only in a second portion of the plurality of micro-fluids that is different from the first portion of the micro-fluids; and performing signal detection on the plurality of micro-fluids to obtain a second signal.

[0140] In one embodiment, the temperature of the plurality of micro-liquids is adjusted to t2, and a first signal is obtained by detecting the plurality of micro-liquids; In a state where the temperature of the plurality of micro-liquids is adjusted to t3, a signal is detected for the plurality of micro-liquids to obtain a second signal; However, t2 <t3である。

[0141] In some embodiments, the difference in temperature between t2 and t3 is 6°C or more, such as, for example, the difference is 6-46°C, including, for example, the difference is 6-40°C, 6-35°C, 6-30°C, 6-25°C, 6-20°C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C, or 6-7°C.

[0142] In one embodiment thereof, the reagent comprises a first reagent and a second reagent; When the temperature of the micro-fluid in the reaction liquid storage section is t2, the first reagent in the micro-fluid in the first portion combines with the sample to generate a signal; When the temperature of the micro-fluid in the reaction liquid storage section is t3, the second reagent in the micro-fluid in the second section combines with the sample to generate a signal.

[0143] In one embodiment thereof, the first temperature regulation mode includes an incubation phase and a signal collection phase; In the incubation stage, the temperature of the plurality of micro-fluids is adjusted so that the plurality of micro-fluids are repeatedly lowered from a high temperature to a low temperature T2, or the temperature of the plurality of micro-fluids is adjusted so that the plurality of micro-fluids maintain a constant temperature T2; In the signal collecting step, signal detection is performed on the multiple micro-fluids to obtain a first signal.

[0144] In one embodiment thereof, the second temperature regulation mode includes an incubation phase and a signal collection phase; In the incubation stage, the temperature of the plurality of micro-fluids is adjusted so that the plurality of micro-fluids are repeatedly lowered from a high temperature to a low temperature T3, or the temperature of the plurality of micro-fluids is adjusted so that the plurality of micro-fluids maintain a constant temperature T3; In the signal collecting step, a second signal is obtained by detecting the signal from the plurality of micro liquids; However, T2>T3 and t2>T3.

[0145] In some embodiments, the difference in temperature between T2 and T3 is 4°C or more, for example, the difference is 4-40°C, including, for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, or 4-5°C.

[0146] In some embodiments, the difference in temperature between t2 and T3 is 5°C or more, for example, the difference is 5-50°C, including, for example, the difference is 5-40°C, 5-30°C, 5-20°C, 5-18°C, 5-15°C, 5-12°C, 5-10°C, 5-8°C, 5-7°C, or 5-6°C.

[0147] In one embodiment, when the temperature of the plurality of micro-fluids is T2, a first reagent in the first portion of the micro-fluids binds with the sample to generate a signal; When the temperature of the multiple micro-fluids is T3, a first reagent in the first portion of the micro-fluids binds with the sample to generate a signal, and a second reagent in the second portion of the micro-fluids binds with the sample to generate a signal.

[0148] In one embodiment, before adjusting the temperature of the plurality of micro-liquids in the first temperature adjustment mode and the second adjustment mode, The temperature of the plurality of micro-fluids is periodically changed to repeatedly lower the plurality of micro-fluids from a high temperature to a low temperature T1, so that a valid signal is generated only in a fourth portion of the plurality of micro-fluids, the fourth portion of the micro-fluids being different from both the first portion of the micro-fluids and the second portion of the micro-fluids, where T1>T2.

[0149] In some embodiments, the difference in temperature between T1 and T2 is 4°C or more, for example, the difference is 4-40°C, such as, for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, or 4-5°C.

[0150] In one embodiment thereof, the reagent further comprises a fourth reagent, When the temperature of the plurality of microfluids is T1, a fourth reagent in the fourth portion of the microfluids binds with the sample to generate a signal; When the temperature of the plurality of micro-fluids is T2, the fourth reagent in the fourth portion of the micro-fluid combines with the sample to generate a signal, and the first reagent in the first portion of the micro-fluid combines with the sample to generate a signal; When the temperature of the multiple micro-fluids is T3, the fourth reagent in the fourth portion of the micro-fluid combines with the sample to generate a signal, the first reagent in the first portion of the micro-fluid combines with the sample to generate a signal, and the second reagent in the second portion of the micro-fluid combines with the sample to generate a signal.

[0151] In one embodiment, a fourth signal is obtained by performing signal detection on the plurality of micro fluids.

[0152] In one embodiment, in the process of performing signal detection on the plurality of micro liquids to obtain a fourth signal, the temperature of the plurality of micro liquids is adjusted to t1 or maintained at T1; When the temperature of the plurality of micro-fluids is T1 or t1, the fourth reagent in the fourth portion of the micro-fluid remains bound to the sample and continues to generate a signal.

[0153] In one embodiment thereof, at most one unit of sample is dispensed into the microfluidic device in an initial state.

[0154] In one embodiment, the signal is a fluorescent signal, and a camera detects the signal from the plurality of micro liquids.

[0155] The detection method and primer probe composition of the present invention have the following advantages over the prior art. (1) Detection signals do not cross over each other. In the detection method and primer probe composition provided by the present invention, in each signal collection for each fluorescent channel, there is at most one detection signal for the target nucleic acid, so that the detection result is more intuitive and accurate, and the overlapping of fluorescent signals of two or more target nucleic acids resulting in detection errors can be avoided. (2) Multiplex detection: The detection method and primer probe composition provided by the present invention can simultaneously perform two-dimensional analysis of fluorescence channel and melting temperature in a single-tube digital PCR reaction, that is, even if the same fluorescence channel is used, different targets (target nucleic acids) can be detected according to their melting temperature characteristics, or different fluorescence channels can be used to realize the detection of target types that are the product of the number of fluorescence channels and the melting temperature characteristics. (3) Low fluorescence background: By using only one probe for each fluorescence channel, different single-stranded preproducts can be distinguished from the probes by their different melting temperatures, which greatly reduces the fluorescence background in digital PCR reactions and improves the reaction sensitivity, while at the same time reducing the number of fluorescent probes in the reagent, greatly reducing the reagent cost, and is favorable for large-scale popularization. (4) Melting temperature adjustable. The method provided by the present invention utilizes different melting temperatures of secondary amplification products (single-stranded pre-products generated by the amplification of a target nucleic acid and a primer set, which is the "primary amplification product"; and a double-stranded product formed after specific binding of the single-stranded pre-product to a probe, which is the "secondary amplification product") to distinguish between the products. Different signal collection temperatures are adjusted by adjusting the length or sequence composition of the primer signal detection region (h) or by adjusting the position of the primer signal detection region (h) that is reversely complementary to the entire signal detection region (H) of the probe (P), and further by raising or lowering the melting temperature of the double-stranded product formed with the probe (P). (5) Good tolerance: In the primer-probe design method described in the present invention, two parts (first primer and second primer) complementarily pair with the target sequence (target nucleic acid) (the probe does not complementarily pair with the target nucleic acid), whereas in the Taqman hydrolysis probe method, three parts (first primer, second primer, and Taqman probe) must complementarily pair with the target sequence. In contrast, when the target sequence contains many highly mutated regions, such as virus or bacterial genomes, the primer-probe design method described in the present invention has good tolerance and is easy to design. (6) Low sample consumption. The primer probe composition and detection method provided by the present invention consumes little sample, which is particularly suitable for detecting rare samples. It can greatly increase the concentration of the sample added to the detection reaction, thereby improving the detection sensitivity, and the single-tube reaction enables detection by the One-Tube method without the need for subsequent extraction. (7) High sensitivity: The method according to the present invention has very low requirements for the length of the target sequence, and when the target type is short fragmented nucleic acid, such as free nucleic acid, the shorter the length of the target sequence, the higher the sensitivity in detection. (8) Wide range of application. The method described in the present invention can be applied to nucleic acid detection in a wide variety of sample types, including serum samples, plasma samples, whole blood samples, sputum samples, swabs, lavage fluid samples, fresh tissue samples, formalin-fixed paraffin-embedded tissues (FFPE), etc.

[0156] Compared with the prior art, the multiplex time-division nucleic acid detection device and detection method of the present invention have the advantage that it can realize different test nucleic acids generating effective signals at different times, i.e., it can isolate the signals of different test nucleic acids, avoid signal overlap and the occurrence of detection errors, and realize time-division, high-precision detection of multiple nucleic acids. [Brief description of the drawings]

[0157] The description of the drawings is provided for a better understanding of the present invention, constitutes a part of the specification, and is used to interpret the present invention together with the following specific embodiments, but is not intended to limit the present invention. In the drawings: [Figure 1]This is a design principle diagram of one type of primer probe of the present invention (for three types of targets). When the target sequence is not present in the sample or amplification has not been initiated, the probe (P) is reverse-complementary only to the primer anchor region (A') and the probe anchor region (A) of the forward primer (F1 / 2 / 3), and the remaining part of the probe does not interact with any sequence. In this case, the distance between the first detection group and the second detection group modified on the probe (P) is d1. During PCR amplification, annealing occurs at a relatively high first annealing temperature (T1), and the forward primer (F1) and reverse primer (R1) each specifically bind to the target sequence 1 and extend to generate double-stranded pre-products, of which the 5'-end to 3'-end of one single-stranded pre-product (S1) is the probe anchor region (A1), the target sequence, the reverse complementary sequence of the primer signal detection region (h1'), and the reverse complementary sequence of the extension block region (M') in this order. The forward primer (F2) and the reverse primer (R2) each specifically bind to the target sequence 2 and extend to generate a double-stranded pre-product, of which one single-stranded pre-product (S2) has a probe anchor region (A2), a target sequence, and a reverse complementary sequence (h2') of the primer signal detection region from the 5' end to the 3' end, respectively. Optionally, when the forward primer (F3) and the reverse primer (R3) are present, the forward primer (F3) and the reverse primer (R3) each specifically bind to the target sequence 3 and extend to generate a double-stranded pre-product, of which one single-stranded pre-product (S3) has a probe anchor region (A3), a target sequence, and a reverse complementary sequence (h3') of the primer signal detection region from the 5' end to the 3' end, respectively. After the PCR amplification is completed, a first signal collection is performed at a relatively low first signal collection temperature (t1).At this time, the probe signal detection region (H) of the probe (P) is complementary to and paired with the reverse complementary sequence (h1’) of the primer signal detection region of the single-stranded pre-product (S1), and the primer anchor region (A1’) of the probe (P) is complementary to and paired with the probe anchor region (A1) at the 5’ end of the single-stranded pre-product (S1), and a double-stranded product (D1) formed with the probe (P) is obtained. However, the reverse complementary sequence (M’) of the extension block region at the 3’ end of the single-stranded pre-product (S1) is not complementary to any part of the probe (P) or the target sequence. At this time, all or part of the sequence of the probe (P) changes from single-stranded to double-stranded, the distance between the first detection group and the second detection group changes, generating a signal that is detected by the device. At this time, the annealing temperatures of the reverse complementary sequences (h2’) of the primer signal detection regions at the 3’ ends of the single-stranded pre-products (S2) and (h3’) of the primer signal detection regions at the 3’ ends of the single-stranded pre-products (S3) are both lower than the first annealing temperature (T1) and the first signal acquisition temperature (t1), so the probe signal detection region (H) of the probe (P) does not extend complementary to it. After the first signal acquisition is completed at the first signal acquisition temperature (t1), a secondary amplification cycle of the single-stranded pre-product (S2) is performed, and the cycle conditions are denaturation at a high temperature and annealing at a relatively low second annealing temperature (T2) (T2 < T1 and T2 < t1). At this time, the probe signal detection region (H) of the probe (P) is complementary to and paired with the reverse complementary sequence (h2’) of the primer signal detection region at the 3’ end of the single-stranded pre-product (S2), and the primer anchor region (A2’) of the probe is complementary to and paired with the probe anchor region (A2) at the 5’ end of the single-stranded pre-product (S2). The 3’ end of the single-stranded pre-product (S2) continues to extend to the 5’ end of the probe (P), and all or part of the reverse complementary sequence of the probe (P) is obtained. That is, a secondary amplification using the single-stranded pre-product (S2) as a primer and the probe (P) as a template is completed, and an amplified double-stranded product (D2) formed with the probe (P) is obtained. At this time, all or part of the sequence of the probe (P) changes from single-stranded to double-stranded, the distance between the first detection group and the second detection group changes, generating a signal.After the cycle amplification is completed at the second annealing temperature (T2), the temperature is raised to a relatively high second signal acquisition temperature (t2) to perform the second signal acquisition (t2 > t1 and t2 > T1). At this time, the hybrid double-stranded product (D1) formed by the single-stranded pre-product (S1) and the probe (P) cannot form a double-stranded structure because the second signal acquisition temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1), and the annealing temperature of the reverse complementary sequence (h3') of the primer signal detection region at the 3'-end of the single-stranded pre-product (S3) is lower than the second annealing temperature (T2), so it does not extend in a reverse complementary manner with the probe signal detection region (H) of the probe (P). The amplification double-stranded product (D2) formed by the single-stranded pre-product (S2) and the probe (P) forms a double-stranded state, the distance between the first detection group and the second detection group becomes far, the fluorescence resonance energy transfer efficiency is low, the fluorescence signal changes, and it is detected by the device. After the second signal acquisition is completed at the second signal acquisition temperature (t2), a secondary amplification cycle of the single-stranded pre-product (S3) is performed, and the cycle conditions are denaturation at a high temperature and annealing at a lower third annealing temperature (T3) (T3 < T2 and T3 < t2). At this time, the probe signal detection region (H) of the probe (P) and the reverse complementary sequence (h3') of the primer signal detection region at the 3'-end of the single-stranded pre-product (S3) are complementary and paired, the primer anchor region (A3') of the probe is reverse complementary and paired with the probe anchor region (A3) at the 5'-end of the single-stranded pre-product (S3), and the 3'-end of the single-stranded pre-product (S3) continues to extend to the 5'-end of the probe (P) to obtain a reverse complementary sequence of part or all of the probe (P), that is, the secondary amplification with the single-stranded pre-product (S3) as the primer and the probe (P) as the template is completed, and the amplification double-stranded product (D3) formed with the probe (P) is obtained. At this time, all or part of the sequence of the probe (P) changes from single-stranded to double-stranded state, the distance between the first detection group and the second detection group changes, and a signal is generated. After the cycle amplification is completed at the third annealing temperature (T3), the temperature is further raised to a higher third signal acquisition temperature (t3) to perform the second signal acquisition (t3 > t2 and t3 > T2).At this time, the hybrid double-stranded product (D1) formed by the single-stranded pre-product (S1) and the probe (P) cannot form a double-stranded structure because the third signal collection temperature (t3) is higher than the melting temperature of the hybrid double-stranded product (D1). The amplified double-stranded product (D2) formed by the single-stranded pre-product (S2) and the probe (P) cannot form a double-stranded structure because the third signal collection temperature (t3) is higher than the melting temperature of the amplified double-stranded product (D2), and the amplified double-stranded product (D3) formed by the single-stranded pre-product (S3) and the probe (P) has formed a double-stranded state. The distance between the first detection group and the second detection group becomes farther, the efficiency of fluorescence resonance energy transfer is lower, and the fluorescent signal changes and is detected by the instrument. [Figure 2A] FIG. 2 is a signal diagram of the digital PCR detection reaction of Example 1 at the first signal collection temperature (60° C.). [Figure 2B] FIG. 1 is a signal diagram of the digital PCR detection reaction of Example 1 at the second signal collection temperature (78° C.). [Figure 3A] FIG. 13 is a signal diagram of the digital PCR detection reaction of Example 2 at the first signal collection temperature (60° C.). [Figure 3B] FIG. 13 is a signal diagram of the digital PCR detection reaction of Example 2 at the second signal collection temperature (78° C.). [Figure 4A] FIG. 11 is a signal diagram at the first signal collection temperature (60° C.) of the digital PCR detection reaction of Example 3. [Figure 4B] FIG. 13 is a signal diagram of the digital PCR detection reaction of Example 3 at the second signal collection temperature (78° C.). [Figure 5A] FIG. 13 is a signal diagram at the first signal collection temperature (56° C.) of the digital PCR detection reaction of Example 4. [Figure 5B] FIG. 13 is a signal diagram of the digital PCR detection reaction of Example 4 at the second signal collection temperature (72° C.). [Figure 6A] FIG. 11 is a signal diagram at the first signal collection temperature (55° C.) of the digital PCR detection reaction of Example 5. [Figure 6B] FIG. 13 is a signal diagram of the digital PCR detection reaction of Example 5 at the second signal collection temperature (65° C.). [Figure 6C]FIG. 13 is a signal diagram of the digital PCR detection reaction of Example 5 at the third signal collection temperature (73° C.). [Figure 7A] FIG. 1 is a signal diagram at the first signal collection temperature (72° C.) of the digital PCR detection reaction of Example 6, showing positive droplets distinguished from the background. [Figure 7B] FIG. 11 is a signal diagram of the digital PCR detection reaction of Example 6 at the second signal collection temperature (82° C.), and it can be seen from the diagram that all of the droplets are negative. [Figure 8A] This is a signal diagram collected in the VIC fluorescence channel at the first signal collection temperature (60° C.) in this Example 7, showing positive droplets that can be distinguished from the background. [Figure 8B] This is a signal diagram collected in the VIC fluorescence channel at the second signal collection temperature (68°C) in this Example 7. Positive droplets that can be distinguished from the background are visible, and the positions of the positive droplets that appeared at this temperature and channel are different from those of the positive droplets that appeared in the VIC channel at the first signal collection temperature (60°C). [Figure 8C] This is a signal diagram collected in the FAM fluorescence channel at the second signal collection temperature (68°C) in Example 7. Positive droplets that can be distinguished from the background are observed, and the positions of the positive droplets that appeared at this temperature and channel are different from those of the positive droplets that appeared in the VIC channel at the first signal collection temperature (60°C) and the VIC channel at the second signal collection temperature (68°C). [Figure 9A] FIG. 13 is a signal diagram at the first signal collection temperature (60° C.) in Example 8. [Figure 9B] FIG. 13 is a signal diagram at the second signal collection temperature (78° C.) in Example 8. [Figure 10A] FIG. 13 is a signal diagram at the first signal collection temperature (60° C.) in Example 9. [Figure 10B] FIG. 13 is a signal diagram at the second signal collection temperature (78° C.) in Example 9. [Figure 11A] FIG. 13 is a signal diagram at the first signal collection temperature (60° C.) in Example 10. [Figure 11B] FIG. 13 is a signal diagram at the second signal collection temperature (78° C.) in Example 10. [Figure 12A]FIG. 13 is a signal diagram at the first signal collection temperature (60° C.) in Example 11. [Figure 12B] FIG. 13 is a signal diagram at the second signal collection temperature (78° C.) in Example 11. [Figure 13A] FIG. 1 is a signal diagram at the first signal collection temperature (61° C.) in Example 1. [Figure 13B] FIG. 13 is a signal diagram at the second signal collection temperature (68° C.) in Example 12. [Figure 13C] FIG. 15 is a signal diagram at the third signal collection temperature (76° C.) in Example 12. [Figure 14A] 1 is a schematic diagram of a multiplex time-division nucleic acid detection device disclosed in one embodiment of the present application. [Figure 14B] FIG. 13 is a schematic diagram of a multiplex time-division nucleic acid detection device disclosed in yet another embodiment of the present application. [Figure 14C] FIG. 13 is a structural diagram of a well plate comprising a plurality of reaction solution storage portions disclosed in yet another embodiment of the present application. [Figure 15A] FIG. 4 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in an embodiment of the present application. [Figure 15B] FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. [Figure 15C] FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. [Figure 15D] FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. [Figure 16A] FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. [Figure 16B] FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. [Figure 16C]FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. [Figure 16D] FIG. 11 is a temperature change curve diagram showing the temperature of a reaction liquid receiving section by controlling a temperature adjusting section using a controller disclosed in yet another embodiment of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0158] In order to facilitate understanding of the present invention, the present invention will be described in detail below with reference to examples. However, these examples only serve as illustrative examples and are not intended to limit the scope of the present invention.

[0159] First embodiment The present invention will be described in detail below with reference to specific embodiments and examples, so that the advantages and various effects of the present invention will become more apparent. Those skilled in the art should understand that these specific embodiments and examples are for the purpose of explaining the present invention, and do not limit the present invention.

[0160] In the following examples, detection and data analysis were performed using a fully automated digital PCR analysis system and reagent consumables (wherein, in Examples 1-7, Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables were used; in Examples 8-12, Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multifunction machine and reagent consumables were used). The digital PCR analysis system distributes a reaction system including a sample, a primer probe composition and an amplification reagent into four reaction wells for amplification and detection (each reaction well contains multiple droplets, each droplet is a reaction cell), and for the sake of simplicity, the drawings in the following examples provide the droplet result diagram of one of the reaction wells.

[0161] Example 1 (1) The single-detection primer-probe system for detecting EGFR exon 21 L858R mutation in non-small cell lung cancer is shown in Table 1.

[0162] [Table 1]

[0163] In the above primer probe, The forward primer F1 (SEQ ID NO:3) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of F1 is 32 bp, and the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A1), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1 (SEQ ID NO:1), i.e., the primer anchor region (A1').

[0164] The reverse primer R1 (SEQ ID NO:2) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of R1 is 50 bp, and the 1st to 20th bases at its 3' end are the target sequence binding region, the 1st to 5th bases at its 5' end are the extension block region (M), and the 6th to 27th bases at its 5' end are the primer signal detection region (h3), which is the same as the 13th to 34th base sequence at the 5' end of the probe P1 (SEQ ID NO:1).

[0165] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 2.

[0166] [Table 2]

[0167] Sample preparation: Non-small cell lung cancer EGFR exon 21 L858R mutation DNA was used as a positive sample, and a reverse primer (R1) with a signal detection region sequence was used to detect the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence, verifying the detection ability for a single mutation. Pure water was used as a templateless control (NTC).

[0168] Reaction configuration: After the sample preparation was completed, the reaction system was arranged according to the formulation shown in Table 2.

[0169] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, for a total of 45 cycles, 60°C for the first signal collection, 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, for a total of 10 cycles, and 78°C for the second signal collection.

[0170] Data analysis (Data analysis was performed using a D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.): The results were judged by the positive droplets at different signal collection temperatures.

[0171] Figure 2A shows a droplet diagram of a single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation positive sample amplified with reverse primer (R1) at the first signal collection temperature (60°C), showing positive droplets distinct from the background.

[0172] Figure 2B shows droplet diagrams of single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation positive samples amplified by reverse primer (R1) at the second signal collection temperature (78°C), and both are seen to be negative droplets.

[0173] As can be seen from Figures 2A and 2B, by adopting the detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures, and the target signal can be turned "on" and "off" to achieve more accurate detection of the target.

[0174] Example 2 (1) The single-detection primer-probe system for detecting EGFR exon 21 L858R mutation in non-small cell lung cancer is shown in Table 3.

[0175] [Table 3]

[0176] In the above primer probe, The forward primer F1 (SEQ ID NO:3) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of F1 is 32 bp, and the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A1), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1 (SEQ ID NO:1), i.e., the primer anchor region (A1').

[0177] The reverse primer R2 (SEQ ID NO: 4) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of R2 is 33 bp, and the 1st to 20th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the primer signal detection region (h2), which is the same as the 1st to 10th base sequence at the 3' end of the probe P1 (SEQ ID NO: 1).

[0178] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 4.

[0179] [Table 4]

[0180] Sample preparation: Non-small cell lung cancer EGFR exon 21 L858R mutation DNA was used as a positive sample, and a reverse primer (R2) with a signal detection region sequence was used to detect the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence, verifying the detection ability for a single mutation. Pure water was used as a templateless control (NTC).

[0181] Reaction configuration: After sample preparation was completed, the reaction system was arranged according to the formulation shown in Table 4.

[0182] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, for a total of 45 cycles, 60°C for the first signal collection, 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, for a total of 10 cycles, and 78°C for the second signal collection.

[0183] Data analysis (Data analysis was performed using a D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.): The results were judged by the positive droplets at different signal collection temperatures.

[0184] Figure 3A shows droplet diagrams at the first signal collection temperature (60°C) of single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation positive samples amplified by the reverse primer (R2), and both are seen to be negative droplets.

[0185] Figure 3B is a droplet diagram of a single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation positive sample amplified with the reverse primer (R2) at the second signal collection temperature (78°C), showing positive droplets distinct from the background.

[0186] As can be seen from Figures 3A and 3B, by adopting the primer probe composition and digital PCR detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures, and the target signal can be turned "off" and "on" to achieve more accurate detection of the target.

[0187] Example 3 (1) The single-detection primer-probe system for detecting EGFR exon 21 L858R mutation in non-small cell lung cancer is shown in Table 5.

[0188] [Table 5]

[0189] In the above primer probe, The forward primer F1 (SEQ ID NO:3) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of F1 is 32 bp, and the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A1), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1 (SEQ ID NO:1), i.e., the primer anchor region (A1').

[0190] The reverse primer R2 (SEQ ID NO: 4) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of R2 is 33 bp, and the 1st to 20th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the primer signal detection region (h2), which is the same as the 1st to 10th base sequence at the 3' end of the probe P1 (SEQ ID NO: 1).

[0191] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 6.

[0192] [Table 6]

[0193] Sample preparation: Non-small cell lung cancer EGFR exon 21 L858R mutation DNA was used as a positive sample, and a reverse primer (R2) with a signal detection region sequence was used to detect the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence, verifying the detection ability for a single mutation. Pure water was used as a templateless control (NTC).

[0194] Reaction configuration: After sample preparation was completed, the reaction system was arranged according to the formulation shown in Table 6.

[0195] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge and centrifuged briefly before being transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 min pre-denaturation, 94°C for 10 s denaturation, 56°C for 30 s annealing extension, a total of 45 cycles, 60°C for the first signal collection, 40°C for 5 min constant temperature incubation, and 78°C for the second signal collection.

[0196] Data analysis (Data analysis was performed using a D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.): The results were judged by the positive droplets at different signal collection temperatures.

[0197] Figure 4A shows droplet diagrams at the first signal collection temperature (60°C) of a single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation positive sample amplified by the reverse primer (R2), all of which are seen to be negative droplets (droplets at the edge and / or appearing relatively large are due to experimental manipulation and are not positive droplets).

[0198] Figure 4B is a droplet diagram of a single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation positive sample amplified with the reverse primer (R2) at the second signal collection temperature (78°C), showing positive droplets distinct from the background.

[0199] As can be seen from Figures 4A and 4B, by adopting the primer probe composition and digital PCR detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures (60°C, 78°C), and the target signal can be turned "off" and "on" to achieve more accurate detection of the target.

[0200] Example 4 (1) The dual detection primer probe system for detecting carbapenem antibiotic resistance gene VIM mutations and carbapenem antibiotic resistance gene OXA-48 mutations is shown in Table 7.

[0201] [Table 7]

[0202] In the above primer probe, The forward primer F1-4 (SEQ ID NO:6) is a specific primer designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The full length of F1-4 is 37 bp, with the 1st to 23rd bases at its 3' end being the target sequence binding region and the 1st to 11th bases at its 5' end being the probe anchor region (A), which is reverse complementary to the 1st to 11th base sequence at the 5' end of the probe P2-4 (SEQ ID NO:5), i.e., the primer anchor region (A').

[0203] The reverse primer R1-4 (SEQ ID NO: 7) is a specific primer designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The total length of R1-4 is 55 bp, with the 1st to 25th bases at its 3' end being the target sequence binding region, the 1st to 5th bases at its 5' end being the signal block region (M), and the 6th to 27th bases at its 5' end being the primer signal detection region (h1), which is the same as the 14th to 35th base sequence at the 5' end of the probe P2-4 (SEQ ID NO: 5).

[0204] The forward primer F2-4 (SEQ ID NO: 8) is a specific primer designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of F2-4 is 33 bp, with the 1st to 19th bases at its 3' end being the target sequence binding region and the 1st to 11th bases at its 5' end being the probe anchor region (A), which is reverse complementary to the 1st to 11th base sequence at the 5' end of the probe P2-4 (SEQ ID NO: 5), i.e., the primer anchor region (A').

[0205] The reverse primer R2-4 (SEQ ID NO: 9) is a specific primer designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of the R2-4 primer is 37 bp, and the 1st to 24th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the same as the 1st to 10th base sequence at the 3' end of the probe P2-4 (SEQ ID NO: 5).

[0206] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 8.

[0207] [Table 8]

[0208] Sample preparation: Carbapenem antibiotic resistance gene VIM mutation nucleic acid and carbapenem antibiotic resistance gene OXA-48 mutation nucleic acid were used as positive samples, and reverse primers (R1-4 and R2-4) with different signal detection region sequences were used to detect carbapenem antibiotic resistance gene VIM mutation and carbapenem antibiotic resistance gene OXA-48 mutation target sequences, and the detection ability for double mutations was verified. Pure water was used as a templateless control (NTC).

[0209] Reaction configuration: After sample preparation was completed, the reaction system was arranged according to the formulation shown in Table 8.

[0210] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, for a total of 45 cycles, 56°C for the first signal collection, 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, for a total of 10 cycles, and 72°C for the second signal collection.

[0211] Data analysis (Data analysis was performed using a D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.): The results were judged by the positive droplets at different signal collection temperatures.

[0212] FIG. 5A is a droplet diagram at the first signal collection temperature (56° C.) in this example, where positive droplets that are distinct from the background are seen and are carbapenem antibiotic resistance gene VIM mutation positive droplets (droplets that are on the edge and / or appear relatively large are due to the experimental manipulation and are not positive droplets).

[0213] FIG. 5B is a droplet diagram at the second signal collection temperature (72°C) in this example, in which positive droplets that can be distinguished from the background are observed, which are carbapenem antibiotic resistance gene OXA-48 mutation positive droplets, and the positive droplets that appeared at this temperature are in a different position than the positive droplets that appeared at the first signal collection temperature (56°C).

[0214] As can be seen from Figures 5A and 5B, by adopting the primer probe composition and digital PCR detection method of the present invention, it is possible to obtain detection signals for different target sequences at different signal collection temperatures (56°C, 72°C), without cross-reacting with each other.

[0215] Example 5 (1) The triple detection primer probe system for detecting the carbapenem antibiotic resistance gene IMP mutation, the carbapenem antibiotic resistance gene OXA-48 mutation, and the carbapenem antibiotic resistance gene KPC mutation is shown in Table 9.

[0216] [Table 9]

[0217] In the above primer probe, The forward primer F1-5 (SEQ ID NO: 11) is a specific primer designed for the carbapenem antibiotic resistance gene IMP mutation target sequence. The total length of F1-5 is 42 bp, and the 1st to 24th bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which is reverse complementary to the 1st to 15th base sequence at the 5' end of probe P1-5 (SEQ ID NO: 10), i.e., the primer anchor region (A').

[0218] The reverse primer R1-5 (SEQ ID NO: 12) is a specific primer designed for the carbapenem antibiotic resistance gene IMP mutation target sequence. The total length of R1-5 is 56 bp, the 1st to 23rd bases at its 3' end are the target sequence binding region, the 1st to 5th bases at its 5' end are the signal block region (M), and the 6th to 30th bases at its 5' end are the primer signal detection region (h1), which is the same as the 17th to 41st base sequence at the 5' end of the probe P1-5 (SEQ ID NO: 10).

[0219] The forward primer F2-5 (SEQ ID NO: 13) is a specific primer designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of F2-5 is 37 bp, and the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which is reverse complementary to the 1st to 15th base sequence at the 5' end of the probe P1-5 (SEQ ID NO: 10), i.e., the primer anchor region (A').

[0220] The reverse primer R2-5 (SEQ ID NO: 14) is a specific primer designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of R2-5 is 38 bp, with the 1st to 24th bases at its 3' end being the target sequence binding region, and the 1st to 11th bases at its 5' end being the same as the 15th to 25th bases at the 3' end of the probe P1-5 (SEQ ID NO: 10).

[0221] The forward primer F3-5 (SEQ ID NO: 15) is a specific primer designed for the carbapenem antibiotic resistance gene KPC mutation target sequence. The total length of F3-5 is 39 bp, and the 1st to 21st bases at its 3' end are the target sequence binding region, and the 1st to 15th bases at its 5' end are the probe anchor region (A), which is reverse complementary to the 1st to 15th base sequence at the 5' end of probe P1-5 (SEQ ID NO: 10), i.e., the primer anchor region (A').

[0222] The reverse primer R3-5 (SEQ ID NO: 16) is a specific primer designed for the carbapenem antibiotic resistance gene KPC mutation target sequence. The total length of R3-5 is 33 bp, and the 1st to 22nd bases at its 3' end are the target sequence binding region, and the 1st to 8th bases at its 5' end are the same as the 1st to 8th base sequence at the 3' end of the probe P1-5 (SEQ ID NO: 10).

[0223] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 10.

[0224] [Table 10]

[0225] Sample preparation: Carbapenem antibiotic resistance gene IMP mutation nucleic acid sample, carbapenem antibiotic resistance gene OXA-48 mutation nucleic acid sample and carbapenem antibiotic resistance gene KPC mutation sample were used as positive samples, and reverse primers (R1-5 / R2-5 / R3-5) with different signal detection region sequences were used to detect carbapenem antibiotic resistance gene IMP mutation, carbapenem antibiotic resistance gene OXA-48 mutation and carbapenem antibiotic resistance gene KPC mutation target sequences, and the detection ability for triple mutations was verified. Pure water was used as a templateless control (NTC).

[0226] Reaction configuration: After sample preparation was completed, the reaction system was arranged according to the formulation shown in Table 10.

[0227] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed uniformly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, a total of 45 cycles, 55°C for the first signal collection, 94°C for 15 seconds denaturation, 52°C for 20 seconds annealing extension, a total of 5 cycles, and 65°C for the second signal collection. 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, a total of 5 cycles, and 73°C for the third signal collection.

[0228] Data analysis (Data analysis was performed using a D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.): The results were judged by the positive droplets at different signal collection temperatures.

[0229] FIG. 6A is a droplet diagram of the carbapenem antibiotic resistance gene IMP mutation, carbapenem antibiotic resistance gene OXA-48 mutation, and carbapenem antibiotic resistance gene KPC mutation positive samples in this example at the first signal collection temperature (55° C.), in which positive droplets that can be distinguished from the background are seen, which are carbapenem antibiotic resistance gene IMP mutation positive droplets.

[0230] FIG. 6B is a droplet diagram of the carbapenem antibiotic resistance gene IMP mutation, carbapenem antibiotic resistance gene OXA-48 mutation, and carbapenem antibiotic resistance gene KPC mutation positive samples in this embodiment at the second signal collection temperature (65°C), in which positive droplets that can be distinguished from the background are observed, which are carbapenem antibiotic resistance gene OXA-48 mutation positive droplets, and the positive droplets that appeared at this temperature are in a different position from the positive droplets that appeared at the first signal collection temperature (55°C).

[0231] FIG. 6C is a droplet diagram of the carbapenem antibiotic resistance gene IMP mutation, carbapenem antibiotic resistance gene OXA-48 mutation, and carbapenem antibiotic resistance gene KPC mutation positive samples in this embodiment at the third signal collection temperature (73°C), which are carbapenem antibiotic resistance gene KPC mutation positive droplets, and the positive droplets that appeared at this temperature are in a different position from the positive droplets that appeared at the first signal collection temperature (55°C) and the second signal collection temperature (65°C).

[0232] As can be seen from Figures 6A-6C, by adopting the primer probe composition and digital PCR detection method of the present invention, it is possible to obtain detection signals of different target sequences at different signal collection temperatures (55°C, 65°C, 73°C), respectively, without cross-reacting with each other.

[0233] Example 6 (1) A single detection primer probe system for detecting non-small cell lung cancer EGFR exon 21 L858R mutation and non-small cell lung cancer EGFR exon 21 L858R mutation is shown in Table 11.

[0234] [Table 11]

[0235] In the above primer probe, The forward primer F2-6 (SEQ ID NO: 18) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of F2-6 is 32 bp, and the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A2), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1-6 (SEQ ID NO: 5), i.e., the primer anchor region (A2').

[0236] The reverse primer R2-6 (SEQ ID NO: 17) is a specific primer designed for the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence. The total length of R2-6 is 33 bp, and the 1st to 20th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the primer signal detection region (h2), which is the same as the 1st to 10th base sequence at the 3' end of the probe P1-6 (SEQ ID NO: 5).

[0237] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 12.

[0238] [Table 12]

[0239] Sample preparation: Non-small cell lung cancer EGFR exon 21 L858R mutation was used as a positive sample, and pure water was used as a template-less control (NTC).

[0240] Reaction configuration: After sample preparation was completed, the reaction system was configured according to the formulation shown in Table 12.

[0241] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, total of 45 cycles, 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, total of 10 cycles, 72°C for the first signal collection, and 82°C for the second signal collection.

[0242] Data analysis (Data analysis was performed using a D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.): The results were determined by the signals collected at different signal collection temperatures.

[0243] FIG. 7A is a droplet view at the first signal collection temperature (72° C.) showing positive droplets that are distinct from the background.

[0244] FIG. 7B is a droplet view at the second signal collection temperature (82° C.), and shows that all droplets are negative.

[0245] As can be seen from Figures 7A and 7B, by adopting the detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures (72°C, 82°C), and the target signal can be turned "on" and "off" to achieve more accurate detection of the target.

[0246] Example 7 (1) The triple detection primer probe system for detecting carbapenem antibiotic resistance gene KPC mutation, carbapenem antibiotic resistance gene IMP mutation, and carbapenem antibiotic resistance gene VIM mutation is shown in Table 13.

[0247] [Table 13]

[0248] In the above primer probe, The forward primer F1-7 (SEQ ID NO: 20) is a specific primer designed for the carbapenem antibiotic resistance gene KPC mutation target sequence. The full length of F1-7 is 36 bp, with the 1st to 21st bases at its 3' end being the target sequence binding region and the 1st to 12th bases at its 5' end being the probe anchor region (A), which is reverse complementary to the 1st to 12th base sequence at the 5' end of the probe P1-7 (SEQ ID NO: 19), i.e., the primer anchor region (A').

[0249] The reverse primer R1-7 (SEQ ID NO: 21) is a specific primer designed for the carbapenem antibiotic resistance gene KPC mutation target sequence. The total length of R1-7 is 52 bp, and the 1st to 22nd bases at its 3' end are the target sequence binding region, the 1st to 5th bases at its 5' end are the signal block region (M), and the 6th to 27th bases at its 5' end are the primer signal detection region (h1), which is the same as the 14th to 35th base sequence at the 5' end of the probe P1-7 (SEQ ID NO: 19).

[0250] The forward primer F2-7 (SEQ ID NO: 22) is a specific primer designed for the carbapenem antibiotic resistance gene IMP mutation target sequence. The total length of F2-7 is 39 bp, and the 1st to 24th bases at its 3' end are the target sequence binding region, and the 1st to 12th bases at its 5' end are the probe anchor region (A), which is reverse complementary to the 1st to 12th base sequence at the 5' end of the probe P1-7 (SEQ ID NO: 19), i.e., the primer anchor region (A').

[0251] The reverse primer R2-7 (SEQ ID NO: 23) is a specific primer designed against the carbapenem antibiotic resistance gene IMP mutation target sequence. The total length of R2-7 is 37 bp, and the 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 11th bases at its 5' end are the same as the 1st to 11th bases at the 3' end of the probe P1-7 (SEQ ID NO: 19).

[0252] The forward primer F3-7 (SEQ ID NO: 24) is a specific primer designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The full length of F3-7 is 36 bp, with the 1st to 23rd bases at its 3' end being the target sequence binding region and the 1st to 10th bases at its 5' end being the probe anchor region (A), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P2-7 (SEQ ID NO: 1), i.e., the primer anchor region (A').

[0253] The reverse primer R3-7 (SEQ ID NO: 19) is a specific primer designed against the carbapenem antibiotic resistance gene VIM mutation target sequence. The total length of R3-7 is 38 bp, with the 1st to 25th bases at its 3' end being the target sequence binding region, and the 1st to 10th bases at its 5' end being the same as the 1st to 10th base sequence at the 3' end of the probe P2-7 (SEQ ID NO: 1).

[0254] The principle of detection using the above primer probe is as follows. During specific amplification of the pathogen target, the forward primer (F1-7) and the reverse primer (R1-7) are annealed at a relatively high first temperature (T1), and each of them specifically binds to the target sequence 1 (carbapenem antibiotic resistance gene KPC mutation) and extends to generate double-stranded pre-amplification products, of which the 5'-end to 3'-end of one single-stranded pre-amplification product (S1) is, in order, the probe anchor region (A1), the target sequence, the reverse complementary sequence of the primer signal detection region (h1'), and the reverse complementary sequence of the extension block region (M'). The forward primer (F2-7) and reverse primer (R2-7) each specifically bind to target sequence 2 (carbapenem antibiotic resistance gene IMP mutation) and extend to generate a double-stranded preamplification product, of which one single-stranded preamplification product (S2) has a probe anchor region (A2), a target sequence, and a reverse complementary sequence (h2') of the primer signal detection region from the 5' end to the 3' end, respectively. The forward primer (F3-7) and reverse primer (R3-7) each specifically bind to target sequence 3 (carbapenem antibiotic resistance gene VIM mutation) and extend to generate a double-stranded preamplification product, of which one single-stranded preamplification product (S3) has a probe anchor region (A3), a target sequence, and a reverse complementary sequence (h3') of the primer signal detection region from the 5' end to the 3' end, respectively.

[0255] After the PCR amplification is completed, signal acquisition is performed at a relatively low first signal acquisition temperature (t1). At this time, the probe signal detection region (H) of the probe (P1-7) is complementary to and paired with the reverse complementary sequence (h1’) of the primer signal detection region of the single-stranded pre-amplification product (S1), and the primer anchor region (A1’) of the probe (P1-7) is complementary to and paired with the probe anchor region (A1) at the 5’ end of the single-stranded pre-amplification product (S1), and a hybrid double-stranded product (D1) formed by the probe (P1-7) is obtained. However, the reverse complementary sequence (M’) of the extension block region at the 3’ end of the single-stranded pre-amplification product (S1) is not complementary to any part of the probe (P1-7) or the target sequence. At this time, a part of the sequence of the probe (P1-7) changes from single-stranded to double-stranded, the distance between the first detection group and the second detection group changes, generating a signal that is detected by the device. At this time, since the annealing temperatures of the reverse complementary sequences (h2’) of the primer signal detection regions at the 3’ ends of the single-stranded pre-amplification products (S2) and (h3’) of the single-stranded pre-amplification product (S3) are both lower than the first annealing temperature (T1) and the first signal acquisition temperature (t1), the probe signal detection region (H) of the probe (P) does not extend complementary to it. After the first signal acquisition is completed at the first signal acquisition temperature (t1), a secondary amplification cycle of the single-stranded pre-amplification products (S2 and S3) is performed, and the cycle conditions are denaturation at a high temperature and annealing at a relatively low second annealing temperature (T2) (T2 < T1 and T2 < t1). At this time, the probe signal detection region (H) of the probe (P1-7) is complementary to and paired with the reverse complementary sequence (h2’) of the primer signal detection region at the 3’ end of the single-stranded pre-amplification product (S2), and the primer anchor region (A2’) of the probe is complementary to and paired with the probe anchor region (A2) at the 5’ end of the single-stranded pre-amplification product (S2). The 3’ end of the single-stranded pre-amplification product (S2) continues to extend to the 5’ end of the probe (P), and all the reverse complementary sequences of the probe (P1-7) are obtained. That is, a secondary amplification with the single-stranded pre-amplification product (S2) as the primer and the probe (P1-7) as the template is completed, and an amplified double-stranded product (D2) formed by the probe (P1-7) is obtained.At this time, a part of the sequence of the probe (P1-7) becomes double-stranded from single-stranded, the distance between the first detection group and the second detection group changes, and a signal is generated. At this time, the probe signal detection region (H) of the probe (P2-7) complementarily pairs with the reverse complementary sequence (h3') of the primer signal detection region at the 3' end of the single-stranded pre-amplification product (S3), the primer anchor region (A3') of the probe complementarily pairs with the probe anchor region (A3) at the 5' end of the single-stranded pre-amplification product (S3), and the 3' end of the single-stranded pre-amplification product (S3) continues to extend to the 5' end of the probe (P2-7), and the entire reverse complementary sequence of the probe (P2-7) is obtained, that is, the single-stranded pre-amplification product (S3) is used as a primer, and the probe (P2-7) is used as a template to complete the secondary amplification, and the amplified double-stranded product (D3) formed with the probe (P2-7) is obtained. At this time, a portion of the sequence of the probe (P2-7) changes from a single-stranded state to a double-stranded state, changing the distance between the first detection group and the second detection group, thereby generating a signal.

[0256] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 14.

[0257] [Table 14]

[0258] Sample preparation: Carbapenem antibiotic resistance gene KPC mutation, gene IMP mutation and gene VIM mutation were used as positive samples, and the three types of samples were mixed and detected to verify the detection ability for triple targets. Pure water was used as a templateless control (NTC).

[0259] Reaction configuration: After sample preparation was completed, the reaction system was configured according to the formulation shown in Table 14.

[0260] Digital PCR amplification and signal collection (detected using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed uniformly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, a total of 45 cycles, 60°C for the first signal collection (VIC channel), 94°C for 15 seconds denaturation, 52°C for 20 seconds annealing extension, a total of 5 cycles, and 68°C for the second signal collection (including two signal channel collections, VIC channel and FAM channel, respectively).

[0261] Data Analysis: The results were judged by positive droplets in different signal channels and at different signal collection temperatures.

[0262] FIG. 8A is a droplet image collected in the VIC fluorescence channel at the first signal collection temperature (60° C.) in this example, in which positive droplets that can be distinguished from the background are seen, which are carbapenem antibiotic resistance gene KPC mutation-positive droplets.

[0263] Figure 8B is an image of droplets collected in the VIC fluorescence channel at the second signal collection temperature (68°C) in this embodiment, in which positive droplets that can be distinguished from the background are observed and are positive for the carbapenem antibiotic resistance gene IMP mutation, and the positive droplets that appeared at this temperature and channel are in a different position than the positive droplets that appeared in the VIC channel at the first signal collection temperature (60°C).

[0264] FIG. 8C is a droplet image collected in the FAM fluorescence channel at the second signal collection temperature (68°C) in this embodiment, in which positive droplets that can be distinguished from the background are observed and are positive droplets for the carbapenem antibiotic resistance gene VIM mutation, and the positive droplets that appeared at this temperature and channel are in a different position than the positive droplets that appeared in the VIC channel at the first signal collection temperature (60°C) and the VIC channel at the second signal collection temperature (68°C).

[0265] As can be seen from Figures 8A to 8C, by adopting the primer probe composition and digital PCR detection method of the present invention, detection signals of different target sequences can be obtained through different signal collection temperatures and different signal collection channels, and do not cross each other, so that the detection results are more intuitive and clearer.

[0266] Example 8 (1) A single-detection primer probe system for detecting non-small cell lung cancer EGFR exon 20 H773_V774insH mutation is shown in Table 15.

[0267] [Table 15]

[0268] In the above primer probe, The forward primer F1-8 (SEQ ID NO: 27) is a specific primer designed for the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence. The full length of F1-8 is 29 bp, and the 1st to 16th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A1), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1-8 (SEQ ID NO: 25), i.e., the primer anchor region (A1').

[0269] The reverse primer R1-8 (SEQ ID NO: 26) is a specific primer designed for the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence. The total length of R1-8 is 51 bp, and the 1st to 21st bases at its 3' end are the target sequence binding region, the 1st to 5th bases at its 5' end are the extension block region (M), and the 6th to 27th bases at its 5' end are the primer signal detection region (h3), which is the same as the 26th to 47th base sequence at the 3' end of the probe P1-8 (SEQ ID NO: 25).

[0270] The principle of detection using the above primer probe is as follows. When specifically amplifying a target, annealing is performed at a relatively high first temperature (T1), and the forward primer (F1-8) and the reverse primer (R1-8) specifically bind to and extend from the target sequences 1 respectively to generate a double-stranded pre-amplification product. Among them, from the 5'-end to the 3'-end of one single-stranded pre-amplification product (S1), there are in sequence a probe anchor region (A1), a target sequence, the reverse complementary sequence (h1') of the primer signal detection region, and the reverse complementary sequence (M') of the extension block region. After the PCR amplification is completed, signal collection is performed at a relatively low first signal collection temperature (t1). At this time, the probe signal detection region (H) of the probe (P1-8) is complementary to and pairs with the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded pre-amplification product (S1), and the primer anchor region (A1') of the probe (P1-8) is complementary to and pairs with the probe anchor region (A1) at the 5'-end of the single-stranded pre-amplification product (S1), obtaining a hybrid double-stranded product (D1) formed by the probe (P1-8). However, the reverse complementary sequence (M') of the extension block region at the 3'-end of the single-stranded pre-amplification product (S1) is not complementary to any part of the probe (P1-8) or the target sequence. At this time, all or part of the sequence of the probe (P) changes from a single-stranded state to a double-stranded state, the distance between the first detection group and the second detection group changes, generating a signal and being detected by the device. After the signal collection is completed at the first signal collection temperature (t1), the cycle conditions further enter a process of denaturing at a high temperature and annealing at a relatively low second annealing temperature (T2) (T2 < T1 and T2 < t1), and signal collection is performed at the second signal collection temperature (t2) (t2 > t1 and t2 > T1). At this time, since the second signal collection temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1) formed by the single-stranded pre-amplification product (S1) and the probe (P1-8), a double-stranded structure cannot be formed, the distance between the first detection group and the second detection group changes, the fluorescence signal changes, and is detected by the device.

[0271] (2) PCR detection is performed using the above primer probe system, and the reaction system used during detection is shown in Table 16.

[0272] [Table 16]

[0273] Sample preparation: Non-small cell lung cancer EGFR exon 20 H773_V774insH mutation DNA was used as a positive sample, and a reverse primer (R1) with a signal detection region sequence was used to detect the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence, and the detection ability for a single mutation was verified. Pure water was used as a templateless control (NTC).

[0274] Reaction configuration: After sample preparation was complete, the reaction system was configured according to the formulation shown in Table 16.

[0275] Digital PCR amplification and signal collection (detected using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function machine and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, for a total of 45 cycles, 60°C for the first signal collection, 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, for a total of 10 cycles, and 78°C for the second signal collection.

[0276] Data analysis (data analysis was performed using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function analyzer analysis software): The results were judged by the positive droplets at different signal collection temperatures.

[0277] Figure 9A is a droplet diagram of a single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation positive sample amplified with reverse primer (R1-8) at the first signal collection temperature (60°C), showing positive droplets distinct from the background.

[0278] Figure 9B shows droplet diagrams at the second signal collection temperature (78°C) of single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation positive samples amplified by reverse primer (R1-8), and both are seen to be negative droplets.

[0279] As can be seen from Figures 9A and 9B, by adopting the primer probe composition and digital PCR detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures, and the target signal can be turned "on" and "off" to achieve more accurate detection of the target.

[0280] Example 9 (1) A single-detection primer probe system for detecting non-small cell lung cancer EGFR exon 20 H773_V774insH mutation is shown in Table 17.

[0281] [Table 17]

[0282] In the above primer probe, The forward primer F1-9 (SEQ ID NO: 27) is a specific primer designed for the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence. The full length of F1-9 is 29 bp, and the 1st to 16th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A1), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1-9 (SEQ ID NO: 25), i.e., the primer anchor region (A1').

[0283] The reverse primer R2-9 (SEQ ID NO: 28) is a specific primer designed for the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence. The total length of R2-9 is 32 bp, and the 1st to 21st bases at its 3' end are the target sequence binding region, and the 1st to 8th bases at its 5' end are the primer signal detection region (h2), which is the same as the 1st to 8th base sequence at the 3' end of the probe P1-9 (SEQ ID NO: 25).

[0284] The principle of detection using the above-mentioned primer probe is as follows. When specifically amplifying the target, annealing is performed at a relatively high first temperature (T1). The forward primer (F1-9) and the reverse primer (R2-9) specifically bind to the target sequence and extend respectively to generate a double-stranded pre-amplification product. Among them, from the 5'-end to the 3'-end of one single-stranded pre-amplification product (S2), in order, there are a probe anchor region (A1), a target sequence, and a reverse complementary sequence (h2') of the primer signal detection region. The first signal collection is performed at a relatively low first signal collection temperature (t1) (t1>T1). Since the annealing temperature of the reverse complementary sequence (h2') of the primer signal detection region at the 3'-end of the single-stranded pre-amplification product (S2) is lower than the first annealing temperature (T1) and the first signal collection temperature (t1), the probe signal detection region (H) of the probe (P1-9) does not extend in a reverse complementary manner. After the first signal collection is completed at the first signal collection temperature (t1), the cycle condition further enters a process of denaturing at a high temperature and annealing at a relatively low second annealing temperature (T2) (T2<T1 and T2<t1). At this time, the probe signal detection region (H) of the probe (P1-9) is complementary to and paired with the reverse complementary sequence (h2') of the primer signal detection region at the 3'-end of the single-stranded pre-amplification product (S2). The primer anchor region (A1') of the probe is reverse complementary to and paired with the probe anchor region (A1) at the 5'-end of the single-stranded pre-amplification product (S2). The 3'-end of the single-stranded pre-amplification product (S2) continues to extend to the 5'-end of the probe (P1-9), and a reverse complementary sequence of the probe (P1-9) is obtained. That is, a secondary amplification with the single-stranded pre-amplification product (S2) as the primer and the probe (P1-9) as the template is completed, and an amplified double-stranded product (D2) formed with the probe (P1-9) is obtained. At this time, the temperature is raised to a relatively high second signal collection temperature (t2) to collect the signal (t2>t1 and t2>T1). The amplified double-stranded product (D2) formed by the single-stranded pre-product (S2) and the probe (P1-9) forms a double-stranded state. The distance between the first detection group and the second detection group becomes far, the fluorescence resonance energy transfer efficiency is low, the fluorescence signal changes, and it is detected by the device.

[0285] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 18.

[0286] [Table 18]

[0287] Sample preparation: Non-small cell lung cancer EGFR exon 20 H773_V774insH mutation DNA was used as a positive sample, and a reverse primer (R2-9) with a signal detection region sequence was used to detect the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence, and the detection ability for a single mutation was verified. Pure water was used as a templateless control (NTC).

[0288] Reaction configuration: After sample preparation was complete, the reaction system was configured according to the formulation shown in Table 18.

[0289] Digital PCR amplification and signal collection (detected using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function machine and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, for a total of 45 cycles, 60°C for the first signal collection, 94°C for 15 seconds denaturation, 40°C for 20 seconds annealing extension, for a total of 10 cycles, and 78°C for the second signal collection.

[0290] Data analysis (data analysis was performed using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function analyzer analysis software): The results were judged by the positive droplets at different signal collection temperatures.

[0291] Figure 10A shows the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation positive sample amplified by the reverse primer (R2-9) at the first signal collection temperature (60°C), and it can be seen that both are negative droplets.

[0292] Figure 10B is a droplet view of a single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation positive sample amplified with reverse primer (R2-9) at the second signal collection temperature (78°C), showing positive droplets distinct from the background.

[0293] As can be seen from Figures 10A and 10B, by adopting the primer probe composition and digital PCR detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures, and the target signal can be turned "off" and "on", thereby realizing more accurate detection of the target.

[0294] Example 10 (1) A single-detection primer probe system for detecting non-small cell lung cancer EGFR exon 20 H773_V774insH mutation is shown in Table 19.

[0295] [Table 19]

[0296] In the above primer probe, The forward primer F1-10 (SEQ ID NO: 27) is a specific primer designed for the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence. The full length of F1-10 is 29 bp, and the 1st to 16th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A1), which is reverse complementary to the 1st to 10th base sequence at the 5' end of the probe P1-10 (SEQ ID NO: 25), i.e., the primer anchor region (A1').

[0297] The reverse primer R2-10 (SEQ ID NO: 28) is a specific primer designed for the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence. The total length of R2-10 is 32 bp, and the 1st to 21st bases at its 3' end are the target sequence binding region, and the 1st to 8th bases at its 5' end are the primer signal detection region (h2), which is the same as the 1st to 8th base sequence at the 3' end of the probe P1-10 (SEQ ID NO: 25).

[0298] The principle of detection using the above-mentioned primer probe is as follows. When specifically amplifying the target, annealing is performed at a relatively high first temperature (T1). The forward primer (F1-10) and the reverse primer (R2-10) specifically bind to and extend from the target sequences 2 respectively to generate a double-stranded pre-amplification product. Among them, from the 5'-end to the 3'-end of one single-stranded pre-amplification product (S3), there are in sequence a probe anchor region (A1), a target sequence, and the reverse complementary sequence (h2') of the primer signal detection region. The first signal acquisition is performed at a relatively low first signal acquisition temperature (t1) (t1 > T1). Since the annealing temperature of the reverse complementary sequence (h2') of the primer signal detection region at the 3'-end of the single-stranded pre-amplification product (S3) is lower than the first annealing temperature (T1) and the first signal acquisition temperature (t1), the probe signal detection region (H) of the probe (P1-10) does not extend in a reverse complementary manner. After the first signal acquisition is completed at the first signal acquisition temperature (t1), the cycle conditions further enter a process of constant-temperature amplification and annealing at a relatively low second annealing temperature (T2) (T2 < T1 and T2 < t1). At this time, the probe signal detection region (H) of the probe (P1-10) and the reverse complementary sequence (h2') of the primer signal detection region at the 3'-end of the single-stranded pre-amplification product (S3) are complementary and paired. The primer anchor region (A1') of the probe is reverse complementary and paired with the probe anchor region (A1) at the 5'-end of the single-stranded pre-amplification product (S3). The 3'-end of the single-stranded pre-amplification product (S3) continues to extend to the 5'-end of the probe (P1), and the reverse complementary sequence of the probe (P1-10) is obtained. That is, a secondary amplification with the single-stranded pre-amplification product (S3) as the primer and the probe (P1-10) as the template is completed, and an amplified double-stranded product (D3) formed with the probe (P1-10) is obtained. At this time, the temperature is raised to a relatively high second signal acquisition temperature (t2) to acquire the signal (t2 > t1 and t2 > T1). The amplified double-stranded product (D3) formed by the single-stranded pre-product (S3) and the probe (P) forms a double-stranded state. The distance between the first detection group and the second detection group becomes far, the fluorescence resonance energy transfer efficiency is low, the fluorescence signal changes, and it is detected by the device.

[0299] (2) PCR detection was performed using the above primer probe system, and the reaction system used for detection is shown in Table 20.

[0300] [Table 20]

[0301] Sample preparation: Non-small cell lung cancer EGFR exon 20 H773_V774insH mutation DNA was used as a positive sample, and a reverse primer (R2-10) with a signal detection region sequence was used to detect the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence, and the detection ability for a single mutation was verified. Pure water was used as a templateless control (NTC).

[0302] Reaction configuration: After sample preparation was complete, the reaction system was configured according to the formulation shown in Table 20.

[0303] Digital PCR amplification and signal collection (detected using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function machine and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge and centrifuged briefly, then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was 95°C for 2 minutes pre-denaturation, 94°C for 10 seconds denaturation, 56°C for 30 seconds annealing extension, a total of 45 cycles, 60°C for the first signal collection, 40°C for 5 minutes incubation, and 78°C for the second signal collection.

[0304] Data analysis (data analysis was performed using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function analyzer analysis software): The results were judged by the positive droplets at different signal collection temperatures.

[0305] FIG. 11A shows droplet diagrams at the first signal collection temperature (60° C.) of a single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation positive sample amplified with reverse primer (R2-10), all of which are seen to be negative droplets (droplets at the edge and / or appearing relatively large are due to experimental manipulation and are not positive droplets).

[0306] FIG. 11B is a droplet view of a single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation positive sample amplified with reverse primer (R2-10) at the second signal collection temperature (78°C), showing positive droplets distinct from the background.

[0307] As can be seen from Figures 11A and 11B, by adopting the primer probe composition and digital PCR detection method of the present invention, different detection signal results can be obtained at different signal collection temperatures, and the target signal can be turned "off" and "on", thereby achieving more accurate detection of the target.

[0308] Example 11 (1) The dual detection primer probe system for detecting influenza A virus (IAV) and respiratory syncytial virus (RSV) is shown in Table 21.

[0309] [Table 21]

[0310] In the above primer probe, The forward primer F1-11 (SEQ ID NO:30) is a specific primer designed for the RS virus target sequence. The full length of F1-11 is 42 bp. The 1st to 29th bases at its 3' end are the target sequence binding region, and the 1st to 10th bases at its 5' end are the probe anchor region (A), which is reverse complementary to the 1st to 10th base sequence at the 5' end of probe P1-11 (SEQ ID NO:25), i.e., the primer anchor region (A').

[0311] The reverse primer R1-11 (SEQ ID NO: 29) is a specific primer designed for the RS virus target sequence. The total length of R1-11 is 54 bp. The 1st to 24th bases at its 3' end are the target sequence binding region, the 1st to 6th bases at its 5' end are the signal block region (M), and the 7th to 27th bases at its 5' end are the primer signal detection region (h1). This is the same as the 26th to 46th base sequence at the 3' end of the probe P1-11 (SEQ ID NO: 25).

[0312] The forward primer F2-11 (SEQ ID NO: 31) is a specific primer designed for the influenza A virus target sequence. The total length of F2-11 is 32 bp. The 1st to 20th bases at the 3' end are the target sequence binding region, and the 1st to 10th bases at the 5' end are the probe anchor region (A), which is reverse complementary to the 1st to 10th base sequence at the 5' end of probe P1-11 (SEQ ID NO: 25), i.e., the primer anchor region (A').

[0313] The reverse primer R2-11 (SEQ ID NO: 32) is a specific primer designed for the influenza A virus target sequence. The total length of the R2-11 primer is 31 bp. The 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 8th bases at its 5' end are the same as the 1st to 8th bases at the 3' end of the probe P1-11 (SEQ ID NO: 25).

[0314] The principle of detection using the above primer probe is as follows: When the target is specifically amplified, annealing is performed at a relatively high first temperature (T1), and the forward primer (F1-11) and reverse primer (R1-11) are specifically bound to target sequence 1 (RS virus) and extended to generate double-stranded pre-amplification products, of which the 5'-end to 3'-end of one single-stranded pre-amplification product (S1) is the probe anchor region (A1), the target sequence, the reverse complementary sequence of the primer signal detection region (h1'), and the reverse complementary sequence of the extension block region (M'). The forward primer (F2-11) and reverse primer (R2-11) are specifically bound to target sequence 2 (influenza A virus) and extended to generate double-stranded pre-amplification products, of which the 5'-end to 3'-end of one single-stranded pre-amplification product (S2) is the probe anchor region (A2), the target sequence, and the reverse complementary sequence of the primer signal detection region (h2'). After the PCR amplification is completed, signal collection is performed at a relatively low first signal collection temperature (t1). At this time, the probe signal detection region (H) of the probe (P1-11) is reverse-complementarily paired with the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded pre-amplification product (S1), and the primer anchor region (A1') of the probe (P1-11) is reverse-complementarily paired with the probe anchor region (A1) at the 5' end of the single-stranded pre-amplification product (S1), resulting in a hybrid double-stranded product (D1) formed with the probe (P1-11), but the reverse complementary sequence (M') of the extension block region at the 3' end of the single-stranded pre-amplification product (S1) is not complementary to any part of the probe (P1-11) or the target sequence. At this time, a part of the sequence of the probe (P1-11) becomes double-stranded from single-stranded, changing the distance between the first detection group and the second detection group, generating a signal that is detected by the instrument. At this time, since the annealing temperature of the reverse complementary sequence (h2') of the primer signal detection region at the 3' end of the single-stranded pre-amplification product (S2) is lower than the first annealing temperature (T1) and the first signal collection temperature (t1), it does not extend in a reverse complementary manner with the probe signal detection region (H) of the probe (P1-11).After completing the first signal acquisition at the first signal acquisition temperature (t1), perform a secondary amplification cycle of the single-stranded pre-amplified product (S2). The cycle conditions are denaturation at a high temperature and annealing at a relatively low second annealing temperature (T2) (T2 < T1 and T2 < t1). At this time, the probe signal detection region (H) of the probe (P1-11) is complementarily paired with the reverse complementary sequence (h2') of the primer signal detection region at the 3' end of the single-stranded pre-amplified product (S2). The primer anchor region (A2') of the probe is reverse complementarily paired with the probe anchor region (A2) at the 5' end of the single-stranded pre-amplified product (S2). The 3' end of the single-stranded pre-amplified product (S2) continues to extend to the 5' end of the probe (P1-11), and the entire reverse complementary sequence of the probe (P1-11) is obtained. That is, a secondary amplification using the single-stranded pre-amplified product (S2) as a primer and the probe (P1-11) as a template is completed, and an amplified double-stranded product (D2) formed with the probe (P1-11) is obtained. At this time, raise the temperature to a relatively high second signal acquisition temperature (t2) (t2 > t1 and t2 > T1) to perform signal acquisition. The amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P1-11) forms a double-stranded state, the distance between the first detection group and the second detection group becomes far, the fluorescence resonance energy transfer efficiency is low, the fluorescence signal changes, and it is detected by the device. At this time, the hybrid double-stranded product (D1) formed by the single-stranded pre-amplified product (S1) and the probe (P1-11) cannot form a double-stranded structure because the second signal acquisition temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1).

[0315] (2) Perform PCR detection using the above primer-probe system, and Table 22 shows the reaction system used during detection.

[0316]

Table 22

[0317] Sample preparation: Influenza A virus (IAV) and Respiratory Syncytial Virus (RSV) were used as positive samples, and reverse primers (R1-11 and R2-11) with different signal detection region sequences were used to detect the Influenza A virus (IAV) and Respiratory Syncytial Virus (RSV) target sequences, verifying the detection ability for dual virus infection. Pure water was used as a templateless control (NTC).

[0318] Reaction configuration: After sample preparation was complete, the reaction system was configured according to the formulation shown in Table 22.

[0319] Digital PCR amplification and signal collection (detected using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function machine and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was reverse transcription at 55°C for 15 min, pre-denaturation at 95°C for 2 min, denaturation at 94°C for 10 s, annealing extension at 56°C for 30 s, for a total of 45 cycles, first signal collection at 60°C, denaturation at 94°C for 15 s, annealing extension at 40°C for 20 s, for a total of 10 cycles, and second signal collection at 78°C.

[0320] Data analysis (data analysis was performed using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function analyzer analysis software): The results were judged by the positive droplets at different signal collection temperatures.

[0321] FIG. 12A is a droplet diagram of the Influenza A virus (IAV) and Respiratory Syncytial Virus (RSV) positive samples from this example at the first signal collection temperature (60° C.), showing positive droplets that are distinct from the background and are Influenza A virus positive droplets (droplets that are on the edge and / or appear relatively large are due to the experimental manipulation and are not positive droplets).

[0322] FIG. 12B is a droplet diagram of the Influenza A virus (IAV) and Respiratory Syncytial Virus (RSV) positive samples of this embodiment at the second signal collection temperature (78°C), in which positive droplets that are distinct from the background are observed and are RS virus positive droplets, and the positive droplets that appeared at this temperature are in a different position than the positive droplets that appeared at the first signal collection temperature (60°C).

[0323] As can be seen from Figures 12A and 12B, by adopting the primer probe composition and digital PCR detection method of the present invention, it is possible to obtain detection signals of different target sequences at different signal detection temperatures (t1, t2), without crossing over with each other.

[0324] Example 12 (1) The triple detection primer probe system for detecting influenza A virus (IAV), influenza B virus (IBV), and respiratory syncytial virus (RSV) is shown in Table 23.

[0325] [Table 23]

[0326] In the above primer probe, The forward primer F1-12 (SEQ ID NO: 35) is a specific primer designed for the influenza A virus (IAV) target sequence. The full length of F1-12 is 33 bp. The 1st to 20th bases at the 3' end of the primer F1-12 are the target sequence binding region, and the 1st to 10th bases at the 5' end of the primer F1-12 are the probe anchor region (A). The forward primer F1-12 is reverse complementary to the 1st to 10th bases at the 5' end of the probe P1-12 (SEQ ID NO: 33), i.e., the primer anchor region (A').

[0327] The reverse primer R1-12 (SEQ ID NO: 34) is a specific primer designed for the influenza A virus (IAV) target sequence. The total length of R1-12 is 47 bp. The 1st to 20th bases at the 3' end are the target sequence binding region, the 1st to 5th bases at the 5' end are the signal block region (M), and the 6th to 24th bases at the 5' end are the primer signal detection region (h1). This is the same as the 30th to 48th base sequence at the 3' end of the probe P1-12 (SEQ ID NO: 33).

[0328] The forward primer F2-12 (SEQ ID NO: 37) is a specific primer designed for the influenza B virus (IBV) target sequence. The full length of F2-12 is 33 bp. The 1st to 20th bases at the 3' end of the primer are the target sequence binding region, and the 1st to 10th bases at the 5' end of the primer are the probe anchor region (A). The primer is reverse-complementary to the 1st to 10th bases at the 5' end of the probe P1-12 (SEQ ID NO: 33), i.e., the primer anchor region (A').

[0329] The reverse primer R2-12 (SEQ ID NO: 36) is a specific primer designed for the influenza B virus (IBV) target sequence. The total length of R2-12 is 35 bp. The 1st to 21st bases at its 3' end are the target sequence binding region, and the 1st to 11th bases at its 5' end are the same as the 17th to 27th bases at the 3' end of the probe P1-12 (SEQ ID NO: 33).

[0330] The forward primer F3-12 (SEQ ID NO: 39) is a specific primer designed for the respiratory syncytial virus (RSV) target sequence. The full length of F3-12 is 35 bp, with the 1st to 22nd bases at its 3' end being the target sequence binding region and the 1st to 10th bases at its 5' end being the probe anchor region (A), which is reverse complementary to the 1st to 10th base sequence at the 5' end of probe P1-12 (SEQ ID NO: 33), i.e., the primer anchor region (A').

[0331] The reverse primer R3-12 (SEQ ID NO: 38) is a specific primer designed for the respiratory syncytial virus (RSV) target sequence. The total length of R3-12 is 29 bp. The 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 7th bases at its 5' end are the same as the 1st to 7th bases at the 3' end of the probe P1-12 (SEQ ID NO: 33).

[0332] The principle of detection using the above primer probe is as follows: When the target is specifically amplified, annealing is performed at a relatively high first temperature (T1), and the forward primer (F1-12) and reverse primer (R1-12) respectively bind specifically to target sequence 1 (RS virus) and extend to generate double-stranded pre-amplification products, of which the 5'-end to 3'-end of one single-stranded pre-amplification product (S1) is the probe anchor region (A1), the target sequence, the reverse complementary sequence of the primer signal detection region (h1'), and the reverse complementary sequence of the extension block region (M'). The forward primer (F2-12) and reverse primer (R2-12) respectively bind specifically to target sequence 2 (influenza A virus) and extend to generate double-stranded pre-amplification products, of which the 5'-end to 3'-end of one single-stranded pre-amplification product (S2) is the probe anchor region (A2), the target sequence, and the reverse complementary sequence of the primer signal detection region (h2'). The forward primer (F3-12) and reverse primer (R3-12) each specifically bind to the target sequence 3 and extend to generate a double-stranded pre-amplification product, of which one single-stranded pre-amplification product (S3) has a probe anchor region (A3), a target sequence, and a reverse complementary sequence (h3') of the primer signal detection region from the 5' end to the 3' end, in that order. After PCR amplification is completed, signal collection is performed at a relatively low first signal collection temperature (t1). At this time, the probe signal detection region (H) of the probe (P1-12) is reverse-complementarily paired with the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded pre-amplification product (S1), and the primer anchor region (A1') of the probe (P1-12) is reverse-complementarily paired with the probe anchor region (A1) at the 5' end of the single-stranded pre-amplification product (S1), forming a hybrid double-stranded product (D1) with the probe (P1-12), but the reverse complementary sequence (M') of the extension block region at the 3' end of the single-stranded pre-amplification product (S1) is not complementary to any part of the probe (P1-12) or the target sequence. At this time, a part of the sequence of the probe (P1-12) becomes double-stranded from single-stranded, changing the distance between the first detection group and the second detection group, generating a signal, which is detected by the instrument.At this time, since the annealing temperatures of the reverse complementary sequences (h2') of the primer signal detection regions at the 3' ends of the single-stranded pre-amplification products (S2) and the reverse complementary sequences (h3') of the primer signal detection regions at the 3' ends of the single-stranded pre-amplification products (S3) are both lower than the first annealing temperature (T1) and the first signal acquisition temperature (t1), the probe signal detection region (H) of the probe (P1-12) does not extend in a reverse complementary manner. After completing the first signal acquisition at the first signal acquisition temperature (t1), a secondary amplification cycle of the single-stranded pre-amplification product (S2) is performed, and the cycle conditions are denaturation at a high temperature and annealing at a relatively low second annealing temperature (T2) (T2 < T1 and T2 < t1). At this time, the probe signal detection region (H) of the probe (P1-12) complementarily pairs with the reverse complementary sequence (h2') of the primer signal detection region at the 3' end of the single-stranded pre-amplification product (S2), and the primer anchor region (A2') of the probe complementarily pairs with the probe anchor region (A2) at the 5' end of the single-stranded pre-amplification product (S2). The 3' end of the single-stranded pre-amplification product (S2) continues to extend to the 5' end of the probe (P1-12), and all the reverse complementary sequences of the probe (P1-12) are obtained. That is, secondary amplification using the single-stranded pre-amplification product (S2) as a primer and the probe (P1-12) as a template is completed, and an amplified double-stranded product (D2) formed with the probe (P1-12) is obtained. At this time, all the sequences of the probe (P1-12) change from single-stranded to double-stranded states, the distance between the first detection group and the second detection group changes, and a signal is generated. At this time, since the second signal acquisition temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1) formed by the single-stranded pre-amplification product (S1) and the probe (P1-12), the double-stranded structure cannot be formed. Also, since the annealing temperature of the reverse complementary sequence (h3') of the primer signal detection region at the 3' end of the single-stranded pre-amplification product (S3) is lower than the second annealing temperature (T2), the probe signal detection region (H) of the probe (P1-12) does not extend in a reverse complementary manner.After completing the second signal acquisition at the second signal acquisition temperature (t2), perform a secondary amplification cycle on the single-stranded pre-amplification product (S3). The cycle conditions are denaturation at a high temperature and annealing at a lower third annealing temperature (T3) (T3 < T2 and T3 < t2). At this time, the probe signal detection region (H) of the probe (P1-12) is complementary to and paired with the reverse complementary sequence (h3') of the primer signal detection region at the 3' end of the single-stranded pre-amplification product (S3). The primer anchor region (A3') of the probe is reverse complementary to and paired with the probe anchor region (A3) at the 5' end of the single-stranded pre-amplification product (S3). The 3' end of the single-stranded pre-amplification product (S3) continues to extend to the 5' end of the probe (P1-12), and a partial or complete reverse complementary sequence of the probe (P1-12) is obtained. That is, a secondary amplification using the single-stranded pre-amplification product (S3) as a primer and the probe (P1-12) as a template is completed, and an amplified double-stranded product (D3) formed with the probe (P1-12) is obtained. At this time, all or part of the sequence of the probe (P1-12) changes from single-stranded to double-stranded, the distance between the first detection group and the second detection group changes, and a signal is generated. After the cycle amplification ends at the third annealing temperature (T3), the temperature is further raised to a higher third signal acquisition temperature (t3) to perform the second signal acquisition (t3 > t2 and t3 > T2). At this time, the hybrid double-stranded product (D1) formed by the single-stranded pre-amplification product (S1) and the probe (P1-12) cannot form a double-stranded structure because the third signal acquisition temperature (t3) is higher than the melting temperature of the hybrid double-stranded product (D1). The amplified double-stranded product (D2) formed by the single-stranded pre-amplification product (S2) and the probe (P1-12) cannot form a double-stranded structure because the third signal acquisition temperature (t3) is higher than the melting temperature of the amplified double-stranded product (D2). The amplified double-stranded product (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P1-12) forms a double-stranded state. The distance between the first detection group and the second detection group becomes longer, the fluorescence resonance energy transfer efficiency is low, the fluorescence signal changes, and it is detected by the device.

[0333] (2) Perform PCR detection using the above primer-probe system, and Table 24 shows the reaction system used during detection.

[0334] [Table 24]

[0335] Sample preparation: Influenza A virus (IAV), Influenza B virus (IBV) and Respiratory Syncytial Virus (RSV) were used as positive samples, and reverse primers (R1-12 / R2-12 / R3-12) with different signal detection region sequences were used to detect the Influenza A virus (IAV), Influenza B virus (IBV) and Respiratory Syncytial Virus (RSV) target sequences, verifying the detection ability for triple virus infection. Pure water was used as a templateless control (NTC).

[0336] Reaction configuration: After sample preparation was complete, the reaction system was configured according to the formulation shown in Table 24.

[0337] Digital PCR amplification and signal collection (detected using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function machine and reagent consumables): After sealing the digital PCR tube with a lid, the sample was gently mixed uniformly, centrifuged briefly, and then left at room temperature for 5 min. The digital PCR tube was again placed in a palmtop centrifuge, centrifuged briefly, and then transferred to the sample holder of the digital PCR device. The amplification and light collection process used was reverse transcription at 55°C for 15 min, pre-denaturation at 95°C for 2 min, denaturation at 94°C for 10 s, annealing extension at 56°C for 30 s, for a total of 45 cycles, first signal collection at 61°C, denaturation at 94°C for 15 s, annealing extension at 52°C for 20 s, for a total of 5 cycles, second signal collection at 68°C. Denaturation at 94°C for 15 s, annealing extension at 40°C for 20 s, for a total of 5 cycles, and third signal collection at 76°C.

[0338] Data analysis (data analysis was performed using Beijing Da Microbiology Technology Co., Ltd.'s OS-500 digital PCR multi-function analyzer analysis software): The results were judged by the positive droplets at different signal collection temperatures.

[0339] FIG. 13A is a droplet diagram of Influenza A virus (IAV), Influenza B virus (IBV), and Respiratory Syncytial Virus (RSV) positive samples in this example at the first signal collection temperature (61° C.), showing positive droplets that are distinct from the background and are Influenza A virus positive droplets.

[0340] FIG. 13B is a droplet diagram of Influenza A virus (IAV), Influenza B virus (IBV), and Respiratory Syncytial Virus (RSV) positive samples from this embodiment at the second signal collection temperature (68°C), in which positive droplets that are distinct from the background are observed, which are Influenza B virus positive droplets, and the positive droplets that appeared at this temperature are in a different position than the positive droplets that appeared at the first signal collection temperature (61°C).

[0341] FIG. 13C is a droplet diagram of Influenza A virus (IAV), Influenza B virus (IBV), and Respiratory Syncytial Virus (RSV) positive samples in this embodiment at the third signal collection temperature (76°C), which is a RS virus positive droplet, and the positive droplets that appeared at this temperature are in a different position than the positive droplets that appeared at the first signal collection temperature (61°C) and the second signal collection temperature (68°C).

[0342] As can be seen from Figures 13A to 13C, by adopting the primer probe composition and digital PCR detection method of the present invention, it is possible to obtain detection signals of different target sequences at different signal detection temperatures (t1, t2, t3), without crossing over with each other.

[0343] The above are only preferred examples of the present invention. Those skilled in the art may make other equivalent modifications and improvements based on the technical teachings provided by the present invention and the common knowledge in the art, which should be considered as within the protection scope of the present invention.

[0344] Second embodiment It should be noted that the terms "comprise" and "have" and any variations thereof in the examples and drawings of the present application are intended to cover non-exclusive inclusions, for example, a process, method, system, product, or device that includes a series of steps or units is not limited to the recited steps or units, but may optionally further include unrecited steps or units, or may optionally further include other steps or units inherent to those processes, methods, products, or devices.

[0345] The terms "first", "second", etc. used in this application may be used to describe various elements in this specification, but it will be understood that these elements are not limited to these terms. These terms are used only to distinguish the first element from other elements. For example, the first mobile information may be referred to as the second mobile information, and similarly, the second mobile information may be referred to as the first mobile information, without departing from the scope of this application. Both the first mobile information and the second mobile information are mobile information, but are not the same mobile information. Furthermore, the term "multiple" used in the examples of this application means two or more than two.

[0346] The technical solutions of the present application are further described below in the form of examples.

[0347] The multiplex time-resolved nucleic acid detection device of this embodiment is for detecting a test sample, which may contain one type of test component, or may contain two, three, four, five or more types of test components, and the multiplex time-resolved nucleic acid detection device of this embodiment can realize multiplex detection of the test components.

[0348] 14A and 14B, the multiplex time-division nucleic acid detection device of this embodiment includes a reaction liquid storage section 1, a temperature control section 2, a signal detection section 3, and a control section 4. Here, the reaction liquid storage section 1 is for storing a test sample containing a test component and a reagent capable of reacting with the test component, and provides a place for causing various reactions between the test sample and the reagent. Here, the temperature control section 2 is for adjusting the temperature of the mixed liquid in the reaction liquid storage section 1 so that the test sample and the reagent cause different reactions at different temperatures. Here, the signal detection section 3 is for detecting a signal due to a specific substance after a specific reaction in the mixed liquid, and the signal may be an optical signal or an electrical signal. Here, the controller 4 is for controlling the temperature control section 2 to adjust the temperature of the mixed liquid in the reaction liquid storage section 1 with different temperature patterns, and for controlling the signal detection section 3 to detect a signal due to a specific substance in the mixed liquid.

[0349] I. Sample The sample is a body fluid from a human or animal body, and the sample includes different test components, which may be nucleic acids or proteins. In this embodiment, the technical solution is described with the test components being nucleic acids. For samples directly collected from a human or animal body, a nucleic acid extraction and purification operation can be performed to extract nucleic acids, which may be performed manually or by a specialized nucleic acid extractor. For samples directly collected from a human or animal body, a nucleic acid extraction and purification operation may not be performed. The sample includes different test nucleic acid molecules, such as a first test component (first test nucleic acid), a second test component (second test nucleic acid), a third test component (third test nucleic acid), a fourth test component (fourth test nucleic acid), a fifth test component (fifth test nucleic acid), etc.

[0350] II. Reagents The reagent includes an amplification substance for amplifying the nucleic acid and a labeling substance for labeling the nucleic acid, the amplification substance can amplify different nucleic acid molecules, and the labeling substance can bind to the nucleic acid at a predetermined temperature to generate a detectable signal. Different nucleic acids have different temperature ranges for binding to the labeling substance and also different temperature ranges for separating from the labeling substance after binding. The primers in the amplification substance for amplifying different test nucleic acids are different, and the labeling substances for labeling different test nucleic acids may be the same or different, and the present application labels different test nucleic acids with the same labeling substance.

[0351] III. Microfluid The microfluid may be a microdroplet formed in the emulsion, or a microfluid contained in a pore on a container. Each microfluid contains at most one unit of nucleic acid molecule and enough reagents to amplify multiple target nucleic acids and label multiple target nucleic acids. For example, if there are two types of test nucleic acids in the test sample, i.e., a first test nucleic acid and a second test nucleic acid, after the micro-partitioning process, each of the first test nucleic acids and each of the second test nucleic acids are distributed into different microfluids, and there is a sufficient amount of reagent in one microfluid, which can not only meet the needs of amplifying and labeling the first test nucleic acid in the microfluid, but also meet the needs of amplifying and labeling the second test nucleic acid in the microfluid.

[0352] The process of preparing the micro-liquid may be a method of forming the micro-liquid by high-frequency vibration. The sample and a sufficient amount of reagent are sucked into the micropipe, the micropipe is inserted under the oily liquid and vibrated at high frequency, and the micro-liquid of the sample and reagent in the micropipe is discharged at a predetermined speed during the vibration process, so that the vibration shakes off the micro-droplets, i.e., the micro-liquid, which is incompatible with the oily liquid and does not react with it. By setting an appropriate vibration frequency, vibration amplitude, and discharge speed of the micro-liquid, it can be ensured that each formed micro-liquid contains at most one unit of nucleic acid molecule.

[0353] The vessel may be provided with a number of microcompartments and the sample and reagent microfluids may be added to the multiple microcompartments such that at most one unit of the nucleic acid molecule is present in each microcompartment.

[0354] IV. Reaction solution storage section As shown in Figure 14A, the reaction liquid storage section is a box-shaped container that opens upward, and the test nucleic acid and reagents can be placed into the box-shaped container by a liquid injection mechanism (i.e., a method of forming a micro-liquid by high-frequency vibration as described above), or the test nucleic acid and reagents can be placed into the box-shaped container by a laboratory worker.

[0355] As shown in FIG. 14B, the reaction liquid storage section is a chamber that communicates with the microchannel, and the test nucleic acid and the reagent are driven to enter the chamber by the microfluidic drive structure.

[0356] In order to realize absolute quantitative detection of the test nucleic acid, the micro-fluid containing the test nucleic acid and the reagent is enclosed in about 20,000 micro-fluids, each of which contains one or no test nucleic acid molecule, and regardless of how many types of test components there are in the test nucleic acid, they are all distributed into different micro-fluids (this is an ideal state, and a small amount of micro-fluid may contain one or more test nucleic acid molecules).

[0357] In this embodiment, the reaction liquid storage section is described as being a box-shaped container, and an oily liquid that is not miscible with the sample or reagent and does not react with the sample or reagent is injected into the reaction liquid storage section, and micro-liquids are formed in the oily liquid using a high-frequency vibration method, and the micro-liquids are spread out on the bottom of the reaction liquid storage section due to their own weight.

[0358] As shown in FIG. 14C, the well plate 100 is made up of a plurality of reaction liquid storage sections 1, the bottom of which is flat and capable of distributing a plurality of micro-liquids on the bottom of the reaction liquid storage section 1.

[0359] V.Temperature control section As shown in FIG. 14A and FIG. 14B, the temperature control unit 2 is provided below, above, or to the side of the reaction liquid storage unit 1, and may provide heat to the reaction liquid storage unit 1 or may absorb heat from the reaction liquid storage unit 1, thereby fulfilling the function of raising and lowering the temperature of the micro-liquid in the reaction liquid storage unit 1. The temperature control unit 2 can change the temperature of the micro-liquid in the reaction liquid storage unit 1 in the range of 4°C to 105°C. As shown in FIG. 14A, when the reaction liquid storage unit 1 is a box-shaped container that opens upward, a dedicated mounting position for mounting the reaction liquid storage unit may be provided in the temperature control unit, and when temperature control of the reaction liquid storage unit 1 is required, the reaction liquid storage unit may be mounted on the mounting position. As shown in FIG. 14B, when the reaction liquid storage unit 1 is a chamber that communicates with a microchannel, the temperature control unit 2 may be installed in an appropriate direction of the reaction liquid storage unit 1 by combining factors such as heating efficiency.

[0360] In this embodiment, the temperature control unit 2 is installed below a well plate 100 consisting of multiple reaction liquid storage sections 1, and by performing heating and cooling operations on the flat well plate 100 in contact with it, the temperature of the micro-liquids in each reaction liquid storage section 1 in the well plate 100 changes as the temperature of the well plate 100 increases or decreases.

[0361] VI. Signal detection section As shown in the figure, the signal detection unit 3 is provided near the reaction liquid storage unit 1, and the signal detection unit may be a device (camera or photoelectric device) that detects an optical signal (e.g., a fluorescent signal) or a device that can detect an electric signal depending on the difference in the signal generated by the micro liquid in the reaction liquid storage unit 1. For example, when the signal detection unit is a camera, it may capture the signals spread in the reaction liquid storage unit. The signal detection unit 3 is movable relative to the reaction liquid storage unit 1, and the control unit controls the drive mechanism to move the signal detection unit 3 to a position close to the reaction liquid storage unit 1 (e.g., upward as shown in FIG. 14A) when it is necessary to detect a signal. As shown in FIG. 14B, the signal detection unit 3 may be fixedly provided relative to the reaction liquid storage unit.

[0362] VII. Control Unit As shown in the figure, the control unit 4 is communicatively connected to the temperature control unit 2 and the signal detection unit 3. The control unit may include a first control unit communicatively connected to the temperature control unit for controlling the temperature control unit to operate in different modes, and a second control unit communicatively connected to the signal detection unit for controlling the signal detection unit to collect signals from the micro liquids in the reaction liquid storage unit (not shown). As shown in Figures 14A and 14B, the control unit 4 may be a single member capable of simultaneously controlling the temperature control unit and the signal detection unit.

[0363] Example 13 The test sample includes a first test component (first test nucleic acid) and a second test component (second test nucleic acid), and a first test-related product (a test nucleic acid undergoes a polymerase chain reaction (PCR) and / or a nucleic acid hybridization and / or a nuclease cleavage process, and a corresponding test-related product is generated; in general, the test-related product is a nucleic acid) generated by the first test nucleic acid can bind to a label in a reagent within a first binding temperature range, and a second test-related product generated by the second test nucleic acid can bind to a label in a reagent within a second binding temperature range, and the maximum temperatures of the first binding temperature range and the second binding temperature range are different. In this embodiment, the maximum temperature of the second binding temperature range is lower than the maximum temperature of the first binding temperature range. For example, the first test-related product can be combined with the labeled substance when the temperature is equal to or lower than A1, and the second test-related product can be combined with the labeled substance when the temperature is equal to or lower than A2, where A2 is lower than A1. The first test-related product can be separated from the labeled substance in the reagent within a first separation temperature range, and the second test-related product can be separated from the labeled substance in the reagent within a second separation temperature range, and the minimum temperatures of the first separation temperature range and the second separation temperature range are different. In this embodiment, the minimum temperature of the second separation temperature range is higher than the minimum temperature of the first separation temperature range. For example, the first test-related product can be separated from the labeled substance when the temperature is equal to or higher than B1, and the second test-related product can be separated from the labeled substance when the temperature is equal to or higher than B2, where B2 is higher than B1.

[0364] In order to realize multiple detection of different test nucleic acids in a sample and to prevent mutual interference of signals between different test nucleic acids, in this embodiment, a mixture of sample and reagents (including amplification substances and labeling substances) is added to a reaction liquid storage section to form multiple micro-liquids, and the controller performs the following control.

[0365] As shown in Figures 15A to 15D, the horizontal axis represents time, the vertical axis represents temperature, and the controller controls the temperature regulator to regulate the temperature of the microfluidic in the reaction solution container to generate a test-related product related to the test nucleic acid in the sample (the test nucleic acid includes a first test nucleic acid and a second test nucleic acid, but exists in different microfluidic). In this process, the microfluidic goes through multiple temperature cycles, and in each temperature cycle, the microfluidic changes to multiple different temperatures and continues at the temperature for a certain period of time, and the number of cycles may be multiple, for example, 20-60, for example, about 40. In each cycle, the microfluidic reaches a high temperature state (temperature t5), the double-stranded nucleic acid becomes a single-stranded nucleic acid, and then the microfluidic reaches a low temperature state (temperature T1), and the single-stranded nucleic acid goes through an annealing and elongation process (annealing and elongation may be performed at different temperatures, and the elongation temperature is higher than the annealing temperature). After multiple temperature cycles, the amount of different test nucleic acids and / or test-related products in the sample increases (i.e., the amount of test nucleic acids and / or test-related products in each microfluid increases, but the type of test nucleic acid remains the same), making it easier to detect. By rationally setting the annealing temperature T1 of the amplification process, the amount of test nucleic acids and / or test-related products increases, but does not bind to the labeling substance in the reagent. The annealing temperature T1 (low temperature T1) can take a value within a first range, but cannot overlap with the range (second range) of the following T2, and the values ​​within the first range are all greater than the values ​​within the second range.

[0366] As shown in Figures 15A to 15D, before performing 40 temperature cycles, the microfluidic solution may be heated to a temperature t0 to pre-denaturate the test nucleic acid in the sample for a certain period of time (about 2 minutes). The pre-denaturation for a certain period of time is for converting the nucleic acid from a double-stranded state to a single-stranded state.

[0367] After the 40 cycles are completed, the controller can control the temperature control unit to adjust the temperature of the micro-liquid in the reaction liquid receiving unit to temperature t1 and maintain it for a certain period of time, and as shown in Figures 15A to 15D, this section is section 1, and the controller can control the signal detection unit to perform one detection operation, and the signal detected this time is a blank signal. The temperature t1 can be higher or lower than the annealing temperature T1, and after the cycles are completed, the temperature control unit can maintain the temperature of the micro-liquid at temperature T1, and the controller controls the signal detection unit to perform one detection operation. Since the signal obtained by the current detection is a blank signal, the current detection operation can be omitted to improve efficiency.

[0368] Next, the controller controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit in the first temperature adjustment mode, and after the adjustment of the first temperature adjustment mode, a first test-related product capable of binding to the labeling substance is generated in the micro-fluid, and the labeling substance bound to the first test-related product generates a signal, and in this state, the controller controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a first signal. Since a single test nucleic acid molecule is distributed into one micro-fluid, that is, one micro-fluid only has one test nucleic acid molecule, and this test nucleic acid molecule may be the first test nucleic acid or may be the second test nucleic acid, and after the adjustment of the first temperature adjustment mode, only the micro-fluid containing the first test nucleic acid can generate a signal, and when multiple micro-fluids overlap, the signal emitted by such micro-fluid is an invalid signal, and the signal emitted by a single micro-fluid that does not overlap is a valid signal, and such micro-fluid is a first portion of the micro-fluid, that is, only the first portion of the micro-fluid among the multiple micro-fluids generates a valid signal. The amplification substance for amplifying the first test nucleic acid in the microfluidic solution and the labeling substance for labeling the first test-related product are the first reagent.

[0369] Specifically, the first temperature regulation mode includes an incubation step and a signal collection step, in which a specific type of test-related product capable of binding to a labeled substance is generated in the incubation step, and some of the specific types of test-related products are decoupled from the labeled substance in the signal collection step, while the remaining test-related product maintains its binding to the labeled substance, thereby completing signal detection in the reaction solution container by the signal detection unit. After incubation through the incubation step in the first temperature regulation mode, when only one type of test-related product binds to the labeled substance, the binding state between that type of test-related product and the labeled substance is maintained in the signal collection step.

[0370] In the incubation stage, in order to realize the binding of the first test-related product with the labeling substance, the temperature control unit periodically changes the temperature of the micro-fluid in the reaction fluid container, so that the reaction fluid container repeatedly drops from a high temperature to a low temperature T2, or the temperature control unit maintains the reaction fluid container at a constant temperature T2, which is lower than the annealing temperature T1. When the temperature of the micro-fluid in the reaction fluid container is T2, the first test-related product in the micro-fluid of the first portion binds with the first reagent to generate a signal. The temperature T2 can take a value within a second range, but cannot overlap with the range (third range) of the following T3, and all values ​​within the second range are greater than values ​​within the third range, and all values ​​within the second range are less than values ​​within the first range.

[0371] In order to increase the binding rate between the test nucleic acid and the labeling substance, as shown in Figures 15A and 15C, in the incubation stage of the first temperature control mode, the control unit controls the temperature control unit to periodically change the temperature of the micro-fluid in the reaction solution container, and in each temperature cycle, the first test related product generated by the first test nucleic acid binds to the labeling substance at T2 and dechains with the labeling substance at a high temperature t5, and after repeating multiple cycles, the amount of the first test related product bound to the labeling substance reaches an ideal maximum value, and the number of cycles is about 15 cycles, for example, 10 cycles. In the last cycle, when the first test related product is bound to the labeling substance, the controller controls the signal detection unit to perform signal detection on the micro-fluid to obtain a first signal. Of course, as shown in Figures 15A and 15C, after the last cycle is completed (i.e., the signal collecting step), the control unit controls the temperature adjusting unit to adjust the temperature of the micro-fluid in the reaction fluid receiving unit to temperature t2 (when temperature t2 has not reached the temperature at which the first test-related product and the labeled substance can be separated / dechained), and maintains the first test-related product and the labeled substance in a combined state for a certain period of time, which period is designated as section 2, and in section 2, the controller may control the signal detecting unit to perform signal detection on the reaction fluid receiving unit to obtain a first signal. The first signal includes a valid signal generated by the micro-fluid in the first portion, and if overlapped micro-fluids exist, it further includes an invalid signal generated by the overlapped micro-fluids, and such invalid signals are eliminated in the subsequent analysis process.

[0372] As shown in Figures 15B and 15D, in the incubation stage of the first temperature adjustment mode, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to be kept at temperature T2, and may continue for a certain period of time to incubate the micro-liquid, which may be 10-1800 seconds. After the incubation time is over, the signal detection unit is controlled to perform signal detection on the reaction liquid storage unit to obtain a first signal. Of course, as shown in Figures 16B and 16D, after the constant temperature incubation is over (signal collection stage), the control unit may control the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to temperature t2, keep the first test nucleic acid and the labeling substance in a combined state, and maintain this for a certain period of time, which is set as section 2, and in section 2, the controller may control the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a first signal.

[0373] Next, the controller controls the temperature control unit to control the temperature of the micro-fluid in the reaction solution container in the second temperature control mode, and after the second temperature control mode is adjusted, only the second test-related product of the pre-amplified product is bound to the labeling substance in the reagent, and the labeling substance bound to the second test-related product generates a signal, and in this state, the controller controls the signal detection unit to perform signal detection on the reaction solution container to obtain a second signal. Similarly, a single nucleic acid molecule is distributed into one micro-fluid, i.e., there is only one nucleic acid molecule in one micro-fluid, and this nucleic acid molecule may be the first test nucleic acid or may be the second test nucleic acid, and after the second temperature control mode is adjusted, only the micro-fluid where the second test nucleic acid is located can generate a signal, and when multiple micro-fluids overlap, the signal emitted by such micro-fluid is an invalid signal, and the signal emitted by a single micro-fluid that does not overlap is a valid signal, and such micro-fluid is the second part of the micro-fluid, i.e., only the second part of the micro-fluid among the multiple micro-fluids generates a valid signal. The second reagent comprises an amplification substance that amplifies the second test nucleic acid in the microfluidic solution, and a labeling substance that labels the second test nucleic acid.

[0374] Specifically, the second temperature control mode includes an incubation step and a signal collection step, in which a specific type of test-related product is bound to the labeled substance in the incubation step, and some of the specific types of test-related products are separated from the labeled substance in the signal collection step, and the remaining test-related product remains bound to the labeled substance, thereby completing signal detection of the reaction liquid storage section by the signal detection section.

[0375] In the incubation stage, in order to realize the binding between the second test-related product and the labeling substance, the temperature control unit periodically changes the temperature of the micro-fluid in the reaction liquid container so that the reaction liquid container repeatedly drops from a high temperature to a low temperature T3, or the temperature control unit maintains the reaction liquid container at a temperature T3 lower than the temperature T2. When the temperature of the micro-fluid in the reaction liquid container is T3, the second test-related product in the second part of the micro-fluid combines with the second reagent to generate a signal, and at the same time, the first test-related product in the micro-fluid containing the first test-related product can also combine with the first reagent to generate a signal. The temperature T3 can take a value within a third range, but cannot overlap with the range (fourth range) that the following T4 can take, and any value within the third range is greater than any value within the fourth range, and any value within the third range is less than any value within the second range.

[0376] In order to improve the binding rate between the test-related product and the labeled substance, as shown in Figures 15A and 15C, in the incubation step of the second temperature control mode, the control unit controls the temperature control unit to periodically change the temperature of the micro-liquid in the reaction liquid storage unit, and in each temperature cycle, the second test-related product generated by the second test nucleic acid binds to the labeled substance at T3 and dechains from the labeled substance at a high temperature t5. After repeating multiple cycles, the amount of the second test-related product binding to the labeled substance reaches an ideal maximum value, and the number of cycles is about 15 cycles, for example, 10 cycles. After the last cycle is completed (i.e., in the signal collecting step), the control unit controls the temperature adjusting unit to adjust the temperature of the micro-fluid in the reaction liquid receiving unit to temperature t3 (temperature t3 reaches the temperature at which the first test-related product and the labeled substance are separated / de-chained, but does not reach the temperature at which the second test-related product and the labeled substance are separated / de-chained), separates / de-chains the first test-related product and the labeled substance (separates / de-chains the first test-related product and the labeled substance in the first portion of the micro-fluid, and the signal generated by the binding disappears), and maintains the second test-related product and the labeled substance in a bound state for a certain period of time, which is designated as period 3. In period 3, the controller controls the signal detecting unit to detect signals in the reaction liquid receiving unit to obtain a second signal. The second signal includes a valid signal generated by the micro-fluid in the second portion, and if overlapped micro-fluids exist, it also includes an invalid signal generated by the overlapped micro-fluids, and such invalid signals are eliminated in the subsequent analysis process.

[0377] Since all values ​​in the third range are smaller than the values ​​in the second range, when the temperature reaches the third temperature range, the first test-related product also binds to the labeled substance. Therefore, in each temperature cycle, both the first test-related product and the second test-related product undergo a process of binding to the labeled substance and separating. After the last cycle, the first test-related product and the labeled substance are dechained before the temperature reaches t3 or exceeds t3 and does not reach t4 (as described below, the temperature t4 reaches a temperature at which the first test-related product and the second test-related product can be separated / dechained from the labeled substance, but does not reach a temperature at which the third test-related product can be separated / dechained from the labeled substance), at which time only the second test-related product binds to the labeled substance, and in this state, the signal detection unit is controlled to perform signal detection on the reaction solution storage unit to obtain a second signal.

[0378] As shown in Figures 15B and 15D, in the incubation step of the second temperature adjustment mode, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to be kept at temperature T3, and may continue for a certain time to incubate the micro-liquid, which may be 10-1800 seconds. After the incubation time is over, the signal detection unit is controlled to perform signal detection on the reaction liquid storage unit to obtain a second signal. At temperature T3, both the first test-related product and the second test-related product are bound to the labeling substance, and after the incubation is over (i.e., in the signal detection step), the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit to temperature t3 and maintain it for a certain time, and at temperature t3, the first test-related product is dechained / separated from the labeling substance. As shown in Figures 15B and 15D, this time period is designated as section 3, in which the first test-related product is separated / de-chained from the labeled substance and maintains the separated / de-chained state, while the second test-related product is still bound to the labeled substance, and in this state (section 3), the controller controls the signal detection unit to perform signal detection for the reaction liquid storage section and obtain a second signal.

[0379] By the above-mentioned control by the controller, when the signal detection unit detects a signal generated from the micro-fluid, only one type of test nucleic acid or its related product can be bound to the labeled substance, and the signals generated from the micro-fluids of two types of test nucleic acids can be completely separated, thereby achieving high-precision dual nucleic acid detection.

[0380] Example 14 The difference between this embodiment and Example 13 is that during the amplification process of the nucleic acid in the sample, binding with the labeling substance in the reagent is simultaneously achieved, improving detection efficiency and improving detection throughput.

[0381] Specifically, the following applies: 16A to 16D, the controller controls the temperature control unit to adjust the temperature of the micro-fluid in the reaction solution container in the first temperature control mode, so that the nucleic acid in the micro-fluid is amplified by the amplification substance in the reagent, the amount of the amplified nucleic acid is increased, and the product is bound to the labeling substance in the reagent. In this process, the micro-fluid undergoes a number of temperature cycles, and in each temperature cycle, the micro-fluid changes to a number of different temperatures and remains at the temperatures for a certain period of time, and the number of cycles is about 40. The annealing temperature of the amplification process is temperature T2, and the annealing temperature reaches a temperature at which the first test-related product (e.g., the amplified product of the first test nucleic acid) can bind to the labeled substance. Thus, the amplification of the test nucleic acid and the binding of the amplified product to the labeled substance are simultaneously realized by adjusting the first temperature control mode. After the amplification is completed, the first portion of the micro-fluid containing the first test nucleic acid can generate a signal. At this time, the controller controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain the first signal. Specifically, after 40 cycles in the first temperature control mode, the controller controls the temperature control unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature t2, so that the first test-related product and the labeled substance are maintained in a bound state, and maintained for a certain period of time. This period is designated as section 2. In section 2, the controller controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain the first signal. Of course, after the cycle is completed, the temperature of the micro-liquid in the reaction liquid storage section may be maintained at T2, and the controller may control the signal detection section to perform signal detection on the reaction liquid storage section to obtain the first signal.

[0382] Next, the controller controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction liquid storage unit in the second temperature adjustment mode, and this control is the same as in Example 13, so the description is omitted.

[0383] Example 15 The differences between this embodiment and the thirteenth or fourteenth embodiment are as follows. First, the sample further includes a third test component (third test nucleic acid), and the third test nucleic acid or the third test related product (the third test nucleic acid generates a corresponding third test related product after undergoing operations such as polymerase chain reaction (PCR) and / or nucleic acid hybridization and / or nuclease cleavage, and in general, the third test related product is a nucleic acid) can bind to a labeling substance in the reagent within a predetermined temperature range, and in this embodiment, the maximum temperature at which the third test nucleic acid or the third test related product binds to the labeling substance is lower than the maximum temperature at which the second test nucleic acid or the second test related product binds to the labeling substance. For example, the third test nucleic acid or the third test related product can bind to the labeling substance at or below temperature A3, which is lower than temperature A2 (the second test nucleic acid or the second test related product can bind to the labeling substance at or below temperature A2). The third test nucleic acid or the third test related product can be separated from the labeled substance in the reagent within a predetermined temperature range, and in this embodiment, the minimum temperature at which the third test nucleic acid or the third test related product separates from the labeled substance is higher than the minimum temperature at which the second test nucleic acid or the second test related product separates from the labeled substance. For example, the third test nucleic acid or the third test related product can be separated from the labeled substance at or above temperature B3, which is higher than temperature B2 (the second test nucleic acid or the second test related product can be separated from the labeled substance at or above temperature B2).

[0384] Secondly, the controller controls the temperature control unit to adjust the temperature of the micro liquid in the reaction liquid storage unit in a second temperature control mode, and after the controller controls the signal detection unit to obtain a second signal, the controller further controls as follows:

[0385] The controller controls the temperature control unit to control the temperature of the micro-fluid in the reaction liquid storage unit in the third temperature control mode, and after the adjustment of the third temperature control mode, only the third test nucleic acid or the third test-related product of the product is bound to the labeling substance in the reagent, and the labeling substance generates a signal through the binding, and in this state, the controller controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a third signal. Similarly, a single nucleic acid molecule is distributed in one micro-fluid, that is, there is only one nucleic acid molecule in one micro-fluid, and this nucleic acid molecule may be the first test nucleic acid, the second test nucleic acid, or the third test nucleic acid, and by adjusting the third temperature control mode, only the micro-fluid where the third test nucleic acid is located can generate a signal, and when multiple micro-fluids overlap, the signal emitted from such a micro-fluid is an invalid signal, and the signal emitted from a single micro-fluid that does not overlap is a valid signal, and such a micro-fluid is a third part of the micro-fluid, that is, only the third part of the micro-fluid among the multiple micro-fluids generates a valid signal. The third reagent comprises an amplification substance that amplifies the third test nucleic acid in the microfluidic solution, and a labeling substance that labels the third test nucleic acid.

[0386] Specifically, the third temperature control mode includes an incubation step and a signal collection step, in which a specific type of test nucleic acid or its corresponding test-related product is bound to a labeled substance in the incubation step, and some of the specific types of test nucleic acid or its corresponding test-related product are separated from the labeled substance in the signal collection step, and the remaining type of test nucleic acid or its corresponding test-related product remains bound to the labeled substance, thereby completing signal detection in the reaction liquid storage section by the signal detection section.

[0387] In the incubation stage, in order to realize the binding of the third test nucleic acid or the corresponding third test-related product with the labeling substance, the temperature control unit periodically changes the temperature of the micro-fluid in the reaction liquid container so that the reaction liquid container repeatedly drops from a high temperature to a low temperature T4, or the temperature control unit maintains the reaction liquid container at a constant temperature T4, and the temperature T4 is lower than the temperature T3. When the temperature of the micro-fluid in the reaction liquid container is T4, the third test nucleic acid or the third test-related product in the micro-fluid containing the third test nucleic acid binds with the third reagent to generate a signal. The temperature T4 can take a value within a fourth range, but cannot overlap with the range (fifth range) that the following T5 (if there is a fourth temperature control mode) can take, and all values ​​within the fourth range are greater than values ​​within the fifth range, and all values ​​within the fourth range are smaller than values ​​within the third range.

[0388] In order to increase the binding rate between the test nucleic acid and the labeled substance, as shown in Figures 15C and 16C, in the incubation stage of the third temperature control mode, the control unit controls the temperature control unit to periodically change the temperature of the micro-liquid in the reaction liquid storage unit, and in each temperature cycle, the third test nucleic acid or the third test related product binds to the labeled substance at a low temperature T4 and dechains / separates from the labeled substance at a high temperature t5. After repeating multiple cycles, the amount of the third test nucleic acid or the third test related product that binds to the labeled substance reaches an ideal maximum value, and the number of cycles is about 15 cycles (e.g., 9 cycles, 10 cycles, etc.). After the final cycle is completed (i.e., in the signal collection step), the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature t4 (temperature t4 reaches a temperature at which the first test nucleic acid (or first test-related product) and the second test nucleic acid (or second test-related product) are separated / dechained from the labeled substance, but does not reach a temperature at which the third test nucleic acid / third test-related product and the labeled substance can be separated / dechained), and the first test nucleic acid (or first test-related product) is separated / dechained from the labeled substance (in the micro-fluid containing the first test nucleic acid, the first test nucleic acid / first test-related product is separated / dechained from the labeled substance). (The second test nucleic acid / second test related product and the labeled substance are separated / de-chained in the microfluid containing the second test nucleic acid, and the signal generated by the binding disappears), the second test nucleic acid / second test related product and the labeled substance are separated / de-chained (The second test nucleic acid / second test related product and the labeled substance are separated / de-chained in the microfluid containing the second test nucleic acid, and the signal generated by the binding disappears), and the third test nucleic acid / third test related product and the labeled substance are maintained in a bound state for a certain period of time, which is designated as section 4, and in section 4, the controller controls the signal detection unit to perform signal detection on the reaction liquid container to obtain a third signal. The third signal includes a valid signal generated by the microfluid in the third portion, and if overlapped microfluids exist, it further includes an invalid signal generated by the overlapped microfluids, and such invalid signals are eliminated in the subsequent analysis process.

[0389] Since all values ​​within the fourth range are smaller than the values ​​within the third range, when the temperature reaches the fourth range, the first test nucleic acid (or the first test related product) and the second test nucleic acid (or the second test related product) also bind to the labeling substance. Thus, in each temperature cycle, the first test nucleic acid (or the first test related product), the second test nucleic acid (or the second test related product) and the third test nucleic acid (or the third test related product) all undergo a process of binding to and separating from the labeling substance. After the final cycle, before the temperature reaches t4 or exceeds t4 and does not reach t5 (when the temperature t4 reaches a temperature at which the first test nucleic acid (or first test related product) and the second test nucleic acid (or second test related product) can be separated / dechained from the labeled substance, but does not reach a temperature at which the third test nucleic acid (or third test related product) can be separated / dechained from the labeled substance), both the first test nucleic acid (or the first test related product) and the second test nucleic acid (or the second test related product) are dechained / separated from the labeled substance, and at this time, only the third test nucleic acid (or the third test related product) remains bound to the labeled substance, and in this state, the signal detection unit is controlled to perform signal detection on the reaction solution storage unit to acquire a third signal.

[0390] 15D, in the incubation stage of the second temperature adjustment mode, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to be kept at temperature T4, and may continue to incubate the micro-fluid for a certain period of time, which may be 10-1000 seconds. After the incubation period is over, the control unit controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a third signal. At temperature T4, the first test nucleic acid (or first test related product), the second test nucleic acid (or second test related product), and the third test nucleic acid (or third test related product) are all bound to the labeled substance, and after the incubation is completed (i.e., in the signal detection step), the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature t4 and maintain it for a certain period of time, and this time period is designated as section 4, as shown in Figures 15D and 16D, and in section 4, the first test nucleic acid (or first test related product) and the second test nucleic acid (or second test related product) are all separated / dechained from the labeled substance and maintain their separated / dechained states, and the third test nucleic acid (or third test related product) is still bound to the labeled substance, and in this state (section 4), the controller controls the signal detection unit to perform signal detection on the reaction liquid storage unit to obtain a third signal.

[0391] By the above-mentioned control by the controller, when the signal detection unit detects a signal generated from the micro-fluid, only one type of test nucleic acid or its related product can be bound to the labeled substance, and the signals generated from the micro-fluids of three types of test nucleic acids can be completely separated, thereby achieving high-precision triplex nucleic acid detection.

[0392] Example 16 The difference between this embodiment and embodiment 13 is that the components contained in the sample are different, so the signal obtained in section 1 of Figures 15A-15D is not a blank signal but a signal due to a microfluid containing a certain test component. In this embodiment, the test component is a fourth test component (fourth test nucleic acid).

[0393] In addition to Example 13, the sample contains a fourth test component (fourth test nucleic acid), the fourth test nucleic acid can be amplified by the amplification substance in the reagent, the generated amplification product (fourth test-related product) can bind to the labeling substance in the reagent, and the labeling substance to which the fourth test nucleic acid amplification product is bound can generate a detectable signal. It should be noted that the amplification of the fourth test nucleic acid and the binding to the labeling substance can be realized within a certain temperature range. In this temperature range, other test nucleic acids, for example, the first test nucleic acid and the second test nucleic acid, can be amplified by the amplification substance in the reagent, but the products cannot bind to the labeling substance in the reagent. For example, the fourth test-related product can bind to the labeling substance at a temperature equal to or lower than A4, and the temperature A4 is higher than the temperature A1 (the first test nucleic acid or its related product can bind to the labeling substance at a temperature equal to or lower than A1). The fourth test nucleic acid or its related product can be separated / dechained from the labeling substance within a certain temperature range, and in this embodiment, the minimum temperature for separation of the fourth test nucleic acid or its related product from the labeling substance is lower than the minimum temperature for separation of the first test nucleic acid or its related product from the labeling substance. For example, the fourth test nucleic acid or its related product can be separated from the labeling substance at a temperature equal to or higher than temperature B4, which is lower than temperature B1.

[0394] At the annealing temperature T1, other test nucleic acids in the sample, such as the first test nucleic acid and the second test nucleic acid, are only amplified in this process without binding to the labeling substance in the reagent, and the fourth test nucleic acid can be amplified and bound to the labeling substance. At the temperature t1, the fourth test nucleic acid or its related product remains bound to the labeling substance, and the signal detected by the signal detection unit is not a blank signal but a signal due to the microfluid containing the fourth test nucleic acid. The temperature t1 may be higher, lower, or equal to the annealing temperature T1.

[0395] The subsequent control process and the differences from Example 13 are as follows. In the process in which the controller controls the temperature adjustment unit to adjust the temperature of the reaction liquid storage unit in the first temperature adjustment mode, when the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature T2, the fourth test nucleic acid or its related product binds to the labeled substance, and the first test nucleic acid or its related product binds to the labeled substance. When the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature t2, the fourth test nucleic acid or its related product is separated / dechained from the labeled substance, and the first test nucleic acid or its related product remains bound to the labeled substance.

[0396] In the process of the controller controlling the temperature adjustment unit to adjust the temperature of the reaction liquid storage unit in the second temperature adjustment mode, when the controller controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature T3, the fourth test nucleic acid or its related product binds to the labeled substance, the first test nucleic acid or its related product binds to the labeled substance, and the second test nucleic acid or its related product binds to the labeled substance. When the controller controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit to temperature t3, the fourth test nucleic acid or its related product and the first test nucleic acid or its related product are both separated / dechained from the labeled substance, and the second test nucleic acid or its related product remains bound to the labeled substance.

[0397] The other control processes are the same as those in the thirteenth embodiment, and therefore the description thereof will be omitted here.

[0398] The temperatures t1, t2, t3, t4, and t5 in the above-mentioned Examples 13 to 16 can be understood as follows. At temperature t1, if the sample contains a fourth test nucleic acid, the fourth test nucleic acid or its related product can remain bound to the labeled substance in the reagent; if the sample does not contain the fourth test nucleic acid, there is no binding between the test nucleic acid or its related product and the labeled substance in the reagent. At temperature t2, only the first test nucleic acid or its related product can remain bound to the label in the reagent. At temperature t3, only the second test nucleic acid or its related product can remain bound to the label in the reagent. At temperature t4, only the third test nucleic acid or its related product can remain bound to the label in the reagent. At temperature t5, all of the test nucleic acids or their related products are dechained.

[0399] The fourth reagent, the first reagent, the second reagent, and the third reagent in the above Examples 13 to 16 can be understood as follows. The fourth reagent includes an amplification substance capable of amplifying the fourth test nucleic acid, and a labeling substance capable of labeling the fourth test nucleic acid. The first reagent contains an amplification substance capable of amplifying the first test nucleic acid, and a labeling substance capable of labeling the first test nucleic acid. The second reagent contains an amplification substance capable of amplifying the second test nucleic acid, and a labeling substance capable of labeling the second test nucleic acid. The third reagent contains an amplification substance capable of amplifying the third test nucleic acid, and a labeling substance capable of labeling the third test nucleic acid.

[0400] Here, the primers in the amplification substances for amplifying the fourth, first, second and third test nucleic acids are different, and the remaining components may be the same or different. The labeling substances for labeling the fourth, first, second and third test nucleic acids may be the same or different.

[0401] In the initial state, i.e., before the microfluidic fluid is incubated, one microfluidic fluid contains at most one test nucleic acid (the fourth test nucleic acid, or the first test nucleic acid, or the second test nucleic acid, or the third test nucleic acid) and a sufficient amount of reagents capable of amplifying and labeling all the test nucleic acids.

[0402] Of course, based on the above examples, those skilled in the art can reasonably infer how to use the technical solutions disclosed in this application to achieve high-precision quadruplex nucleic acid detection, quintuplex nucleic acid detection, sextuplex nucleic acid detection, etc.

[0403] Those skilled in the art can understand that the implementation of all or part of the flow of the method of the above embodiments can be completed by instructing relevant hardware by a computer program, and the program can be stored in a non-volatile computer-readable storage medium, and when the program is executed, it can include the flow of each of the above method embodiments. Here, the storage medium can be a magnetic disk, an optical disk, a ROM, etc.

[0404] Any references to memory, storage, database, or other medium used herein may include non-volatile and / or volatile memory. Suitable non-volatile memory may include ROM, Programmable ROM (PROM), Erasable PROM (EPROM), Electrically Erasable PROM (EEPROM), or flash memory. Volatile memory may include random access memory (RAM), used as external cache memory. By way of explanation and not limitation, the RAM may be of various types, such as, for example, static RAM (SRAM), dynamic random access memory (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), enhanced synchronous DRAM (ESDRAM), synchronous link DRAM (SLDRAM), Rambus RAM (RDRAM), and direct Rambus DRAM (DRDRAM).

[0405] It should be understood that "one embodiment" or "one embodiment" throughout the specification means that a particular feature, structure, or characteristic associated with an embodiment is included in at least one embodiment of the present application. Thus, the appearances of "in one embodiment" or "in one embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Those skilled in the art should also understand that any of the embodiments described herein are alternative embodiments, and that the associated operations and modules are not necessarily essential to the present application.

[0406] In various embodiments of the present application, it should be understood that the magnitude of the numbers of each of the above processes does not necessarily mean the order of execution, and the order of execution of each process should be determined by the function and internal logic of the process, without limiting the implementation process of the embodiments of the present application.

[0407] Each functional unit in each embodiment of the present application may be integrated into one processing unit, each unit may exist physically alone, or two or more units may be integrated into one unit. The integrated unit may be realized in the form of hardware or in the form of a software functional unit.

[0408] When the above integrated unit is realized in the form of a software functional unit and sold or used as an independent product, it may be stored in a computer-accessible memory. Based on this understanding, the technical solution of the present application may be essentially or in part contributing to the prior art, or the whole or part of the technical solution may be embodied in the form of a software product, and the computer software product is stored in a memory and includes some requirements for causing a computer device (which may be a personal computer, a server, or a network device, etc., specifically, a processor of a computer device) to perform some or all of the steps of the above-mentioned method in each embodiment of the present application.

[0409] The multiplex time-division nucleic acid detection device and detection method disclosed in the examples of the present application have been described in detail above. Although the principles and embodiments of the present application have been described in this specification using specific examples, the description of the above examples is merely intended to aid in the understanding of the method and its central idea of ​​the present application. At the same time, those skilled in the art may have changes in the specific embodiments and application scope based on the idea of ​​the present application, and as such, the contents of this specification should not be understood as limiting the present application.

Claims

1. A step S100 of mixing a primer probe composition, a test sample, and an amplification reagent containing DNA polymerase and dNTPs to obtain a reaction system; Step S200: performing a first PCR amplification on the reaction system, the annealing temperature used during the amplification being T1; Step S300: performing any one of the following operations; S310: performing a first signal collection at a first signal collection temperature t1; Step 1, comprising: performing a second PCR amplification, and the annealing temperature used during the amplification is T2; Operation 2 includes S310′ of collecting signals for the first time at a first signal collection temperature t1; S310″, in which a second PCR amplification is performed and the annealing temperature used during amplification is T2; and S320″ performing a first signal collection at a first signal collection temperature t1; The above operation satisfies T2<T1 and T2<t1, and preferably satisfies 40°C≦T2<T1≦75°C and / or 0°C≦t1<t2≦90°C, A step S400 of collecting signals for a second time at a second signal collecting temperature t2, where t2>t1 and t2>T1; wherein the test sample contains one type of test target, A method for detecting a target nucleic acid, wherein a target signal is contained in at most one of the first and second signal collections.

2. The reaction conditions for the first PCR amplification in step S200 include pre-denaturation at about 85°C to about 105°C for about 0 to about 15 minutes, denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 40°C to about 75°C for about 3 to about 90 seconds, and 20 to 60 cycles. Preferably, when the target in the test sample is RNA, the amplification reagent further includes a reverse transcriptase. The reaction conditions for the first PCR amplification in the reaction system include reverse transcription at about 30°C to about 65°C for about 2 to about 30 minutes, pre-denaturation at about 85°C to about 105°C for about 0 to about 15 minutes, denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 40°C to about 75°C for about 3 to about 90 seconds, and 20 to 60 cycles. And / or the method according to claim 1, wherein the second PCR amplification in step S300 includes reaction conditions of denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 3 to about 90 seconds, and 1 to 20 cycles, or incubation at a constant temperature of about 20°C to about 75°C for about 10 to about 1800 seconds.

3. The method according to claim 1 or 2, further comprising distributing the reaction system into 500 or more reaction cells, each reaction cell containing one target nucleic acid of a test sample or no target nucleic acid of a test sample, before performing a first PCR amplification on the reaction system, and preferably, the signal collecting comprises collecting a fluorescent signal by a camera, and the signal channel collecting comprises collecting a fluorescent signal by a camera in a fluorescent signal channel.

4. The method according to claim 1 or 2, characterized in that the primer probe composition comprises a probe (P) and a primer, the primer specifically binds to a target in the test sample and then generates a pre-product, the pre-product comprises one single-stranded pre-product capable of specifically binding to a probe, the single-stranded pre-product specifically binds to the probe and forms a double-stranded product after being extended by ≧0 bases, and the formation of the double-stranded product causes a change in the probe signal, preferably the extension is an extension of 0-100 bases, more preferably an extension of 1-100 bases, and / or the concentration of the primer is about 30 nM to about 1000 nM and the concentration of the probe (P) is about 100 nM to about 1200 nM.

5. The probe (P) is a freely designed sequence that does not bind to any target sequence, includes a probe signal detection region (H), and is modified with a detection label. The primer includes a forward primer (F) and / or a reverse primer (R). The forward primer (F) comprises a target sequence binding region that specifically binds to a target sequence, the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region, or the reverse primer (R) comprises an extension block region (M), a primer signal detection region (h) and a target sequence binding region in this order from the 5' end to the 3' end; wherein the target sequence binding region of the reverse primer (R) is a sequence that specifically binds to a target sequence, the primer signal detection region (h) of the reverse primer (R) is a sequence that does not bind to any target sequence, the complementary sequence (h') of the primer signal detection region (h) specifically binds to the probe signal detection region (H) of the probe (P), and the extension block region (M) of the reverse primer (R) is a freely designed sequence that is neither identical to nor complementary to any part of the sequence of the probe (P) or any target sequence, The method according to claim 4, wherein preferably there is an interval of 0-20 bases between the primer signal detection region (h) and the target sequence binding region in the reverse primer (R).

6. The forward primer (F) further comprises a probe anchor region (A), the probe anchor region (A) in the forward primer (F) being located at the 5' end of the target sequence binding region, wherein the probe anchor region (A) is a sequence that does not pair with and bind to any target sequence, and preferably, there is an interval of 0-20 bases between the probe anchor region (A) and the target sequence binding region in the forward primer (F); The method according to claim 5, characterized in that the probe (P) further comprises a primer anchor region (A'), which is a sequence complementary to the probe anchor region (A) of the forward primer (F), and preferably, there is an interval of 0-20 bases between the primer anchor region (A') and the probe signal detection region (H) in the probe (P).

7. A step of mixing a primer probe composition containing at least one probe (P) and at least two primers for different targets, a test sample, and an amplification reagent containing DNA polymerase and dNTPs to obtain a reaction system; and performing n PCR amplifications and n signal collections on the reaction system, wherein one signal collection is performed after each PCR amplification, each signal collection includes at least one signal channel collection, and each signal channel collection includes at most one target signal; The annealing temperature used in the nth PCR amplification is Tn, the collection temperature used in the nth signal collection is tn, Tn<T(n-1), Tn<t(n-1), tn>t(n-1), and tn>T(n-1), preferably 40°C≦Tn<T(n-1)≦75°C, and / or 0°C≦t(n-1)<tn≦90°C; The n is an integer of ≧2, and the n is ≦ the number of types of test targets in the test sample; Preferably, the at least one signal collection includes m signal channel collections, m being the number of types of detection labels of the probes in the primer probe composition, and the product of n and m being ≧ the number of types of test targets in the test sample.

8. The method according to claim 7, wherein the reaction conditions for carrying out a first PCR amplification on the reaction system include pre-denaturation at about 85°C to about 105°C for about 0 to about 15 minutes, denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 40°C to about 75°C for about 3 to about 90 seconds, and 20 to 60 cycles, preferably, when the target in the test sample is RNA, the amplification reagent further includes a reverse transcriptase, and the reaction conditions for carrying out a first PCR amplification on the reaction system include reverse transcription at about 30 to about 65°C for about 2 to about 30 minutes, pre-denaturation at about 85°C to about 105°C for about 0 to about 15 minutes, denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 40°C to about 75°C for about 3 to about 90 seconds, and 20 to 60 cycles.

9. The reaction conditions for the first PCR amplification of the reaction system are as follows: pre-denaturation at about 85° C. to about 105° C. for about 0 to about 15 minutes, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 40° C. to about 75° C. for about 3 to about 90 seconds, and about 20 to about 60 cycles of denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 2 .... or the reaction conditions for performing a first PCR amplification on the reaction system include pre-denaturation at about 85° C. to about 105° C. for about 0 to about 15 minutes, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 40° C. to about 75° C. for about 3 to about 90 seconds, performing 20 to 60 cycles, and incubating at a constant temperature of about 20° C. to about 75° C. for about 10 to about 1800 seconds; Preferably, when the target in the test sample is RNA, the amplification reagent further comprises a reverse transcriptase, and the reaction conditions for the first PCR amplification in the reaction system are reverse transcription at about 30 to about 65° C. for about 2 to about 30 minutes, pre-denaturation at about 85° C. to about 105° C. for about 0 to about 15 minutes, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 40° C. to about 75° C. for about 3 to about 90 seconds, 20 to 60 cycles, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 20° C. to about 75° C. for about 3 to about 90 seconds, and annealing and extension at about 20° C. to about 75° C. for about 3 to about 90 seconds.

8. The method of claim 7, wherein the reaction conditions for a first PCR amplification of the reaction system include reverse transcription at about 30° C. to about 65° C. for about 2 to about 30 minutes, pre-denaturation at about 85° C. to about 105° C. for about 0 to about 15 minutes, denaturation at about 85° C. to about 105° C. for about 1 to about 60 seconds, annealing and extension at about 40° C. to about 75° C. for about 3 to about 90 seconds, 20 to 60 cycles, and incubation at about 20° C. to about 75° C. for about 10 to about 1800 seconds.

10. The method according to any one of claims 7 to 9, wherein the other PCR amplification after the first PCR amplification comprises reaction conditions of denaturation at about 85°C to about 105°C for about 1 to about 60 seconds, annealing and extension at about 20°C to about 75°C for about 3 to about 90 seconds, and 1 to 20 cycles, or incubation at a constant temperature of about 20°C to about 75°C for 10 to 1800 seconds.

11. The method according to claim 7, further comprising distributing the reaction system into 500 or more reaction cells, each reaction cell containing one target nucleic acid of a test sample or no target nucleic acid of a test sample, before performing a first PCR amplification on the reaction system, and preferably, the signal collecting comprises collecting a fluorescent signal by a camera, and the signal channel collecting comprises collecting a fluorescent signal by a camera in a fluorescent signal channel.

12. The method according to any one of claims 7 to 11, characterized in that at least one of the primers generates a pre-product after specifically binding to a target, the pre-product comprises one single-stranded pre-product capable of specifically binding to a probe (P), the single-stranded pre-product forms a double-stranded product after specifically binding to the probe (P) and being extended, the formation of the double-stranded product causes a change in the probe signal, preferably the extension is an extension of 0-100 bases, more preferably the extension is an extension of 1-100 bases, and / or the concentration of the primer is about 30 nM to about 1000 nM and the concentration of the probe (P) is about 100 nM to about 1200 nM.

13. The at least two primers for different targets include any one of the following: (1) at least two types of forward primers (F) and at least one type of reverse primer (R); (2) at least one type of forward primer (F) and at least two types of reverse primers (R); (3) at least two types of forward primers (F) and at least two types of reverse primers (R); The different forward primers (F) each independently contain a target sequence binding region that specifically binds to a different target sequence, and / or the probe (P) is a freely designed sequence that does not bind to any target sequence, includes a probe signal detection region (H), and is modified with a detection label; and / or the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region, wherein the target sequence binding regions of the different reverse primers (R) have sequences that specifically bind to different target sequences, and the primer signal detection regions (h) of the different reverse primers (R) do not bother to bind to any target sequence, and the complementary sequence of the primer signal detection region (h) specifically binds to the probe signal detection region (H) of the probe (P), and preferably the sequences of the primer signal detection regions (h) of the different reverse primers (R) are different from each other.

14. The method according to any one of claims 7 to 13, characterized in that, in the primer, at least one type of reverse primer (R) comprises, from the 5'-end to the 3'-end, an extension block region (M), a primer signal detection region (h) and a target sequence binding region, the extension block region (M) is a freely designed sequence that is neither identical nor complementary to any part of the sequence of the probe (P) or any target sequence, and if there is another type of reverse primer, the 5'-end of the other type of reverse primer (R) does not contain the extension block region (M).

15. In the primers, at least one type of forward primer (F) further comprises a probe anchor region (A), the probe anchor region (A) in the forward primer (F) is located at the 5' end of the target sequence binding region, wherein the probe anchor region (A) is a sequence that does not pair with and bind to any target sequence, the sequences of the target sequence binding regions of different forward primers (F) are different, and preferably, there is an interval of 0-20 bases between the probe anchor region (A) and the target sequence binding region in the forward primer (F); The method according to any one of claims 7 to 14, characterized in that the probe (P) is a sequence that does not pair with and bind to any target sequence, and includes a primer anchor region (A') and a probe signal detection region (H), the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of a forward primer (F), and preferably there is an interval of 0 to 20 bases between the primer anchor region (A') and the probe signal detection region (H) in the probe (P).

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