Method for detecting multiple target nucleic acids

Abstract

The present invention relates to the field of molecular biology, specifically to a method for detecting multiple target nucleic acids in a single sample container. In particular, the present invention relates to a method for multiplex detection by digital PCR, which includes mixing a primer probe composition, a test sample, and an amplification reagent to obtain a reaction system, placing the reaction system under conditions that allow hybridization and extension reaction by nucleic acid polymerase, obtaining a reaction product, and collecting signals from the reaction product. The above detection method can realize the detection of multiple target nucleic acids, and is easy to operate and time-saving.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to Chinese patent application CN202111617233.2, entitled "Detection primer probe, primer probe set and application thereof," filed on December 27, 2021, the entire contents of which are incorporated herein by reference.

[0002] [Technical field] The present invention relates to the field of molecular biology, and in particular to a method for detecting multiple target nucleic acids in a single sample vessel. [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 order to realize multiplex detection in digital PCR, the commonly used method is to design a probe for each target, and use different probes with different fluorescence to detect different targets. However, too many probes not only increase the cost, but also increase the fluorescence background and reduce the detection sensitivity. Summary of the Invention

[0007] In order to solve the above problems, the present invention provides mixing the primer probe composition, a test sample, and an amplification reagent containing a nucleic acid polymerase and dNTPs to obtain a reaction system; placing the reaction system under conditions that allow hybridization and extension reactions by a nucleic acid polymerase to obtain a reaction product; The reaction product is placed at n different signal collection temperatures to perform a total of n signal collections, and one signal collection is performed at each of the signal collection temperatures, and each signal collection includes at least one signal channel collection; Analyzing whether there is a difference between signals obtained in two adjacent collections of the same signal channel to determine whether a target nucleic acid is present in the test sample; and / or Analyzing the presence or absence of a signal obtained in the same signal channel at the highest signal collection temperature employed to identify whether or not the target nucleic acid is present in the test sample; The present invention provides a method for detecting multiple types of target nucleic acids, wherein n is an integer of ≧2 and n≦the number of types of target nucleic acids.

[0008] According to the above detection method, it is possible to realize detection of multiple kinds of target nucleic acids (at least 8-plex reaction can be realized in digital PCR). The realization of multiplex detection is not only dependent on the fluorescence channel, but also utilizes different melting temperatures in the same fluorescence channel (i.e., the same signal channel), and by distinguishing the presence or absence of fluorescence through a limited number of photographs, different targets can be distinguished, which reduces the requirements for fluorescence discrimination in digital PCR, simplifies the operation, and saves time.

[0009] In some embodiments, one signal collection is performed at each signal collection temperature, and when n different signal collection temperatures are used, n signal collections are performed. In some embodiments, n is the number of signal collections, and n≦the number of types of target nucleic acids, i.e., the number of signal collections≦the number of types of target nucleic acids.

[0010] In some embodiments, the number of types of target nucleic acids contained in the signals obtained in two adjacent signal channel collections in the same signal channel differs by at most one type. In some embodiments, the number of types of target nucleic acids contained in the signals obtained in two adjacent signal channel collections in the same signal channel differs by at most one type. In some embodiments, the signal obtained in the signal channel collection using the highest signal collection temperature includes at most one type of target nucleic acid. In some embodiments, the "one type of target nucleic acid" further includes a class 1 target nucleic acid that is not classified.

[0011] In some embodiments, "signal collection" includes the operation of performing a first signal collection of the reaction product at a first temperature, then increasing the temperature to a second temperature and performing a second signal collection, then continuing to increase the temperature to a third temperature and performing a third signal collection, and then continuing to increase the temperature and collect signals until all of the target nucleic acid in the test sample is detected. For example, it may include the operation of performing a first signal collection of the reaction product at a first temperature, then decreasing the temperature to a second temperature and performing a second signal collection, then continuing to decrease the temperature to a third temperature and performing a third signal collection, and then continuing to decrease the temperature and collect signals until all of the target nucleic acid in the test sample is detected, and collecting signals during continuous temperature decrease.

[0012] In some embodiments, the primer probe composition has only one type of probe (one type of probe refers to one type of probe with one type of modified detection label or fluorescent group, which may be the same or different in base sequence), and each signal collection includes only one signal channel collection, that is, each signal collection is performed once in a certain signal channel (fluorescence channel). In this case, collection of two adjacent signal channels is two adjacent signal collections, and if the number of types of target nucleic acids contained in the signals obtained by collecting two adjacent signal channels differs by one, the number of signal collections n is equal to the number of types of target nucleic acids. For example, a test sample contains three types of target nucleic acids, and a reaction product is collected in a first signal collection (corresponding to signal channel collection) in a certain fluorescence channel at a first temperature, and the obtained signal contains three types of targets, and then the temperature is raised to a second temperature and a second signal collection is performed, and the obtained signal contains two types of targets, and then the temperature is raised to a third temperature and a third signal collection is performed, and the obtained signal contains one type of target. By comparing the signal status of two adjacent signal collections, the presence status of target nucleic acid can be identified.For example, when a part of the signal disappears during the second signal collection compared to the first signal collection, the disappeared signal is the first target, when another part of the signal disappears during the third signal collection compared to the second signal collection, the other part of the disappeared signal is the second target, and the signal still present during the third signal collection is the third target, thus, it is possible to identify whether or not all three types of targets are present.In addition, the amount of each target can be identified according to the number of disappearances / presences of signals.

[0013] In some embodiments, when the primer probe composition has two kinds of probes (two kinds of probes refer to two kinds of probes with different modified detection labels or fluorescent groups and different base sequences), at least one signal collection includes two signal channel collections. Here, two signal channel collections, that is, one signal collection is performed in each of two signal channels (fluorescence channels). Those skilled in the art can predict the melting temperature of the reaction product, and thus the temperature required for the reaction product representing the target nucleic acid to generate a detectable signal, and the fluorescence channel in which it is located, according to the design situation of the primer probe composition. Therefore, those skilled in the art can select an appropriate signal collection temperature and perform signal collection in an appropriate signal channel (fluorescence channel) according to the design situation of the primer probe composition. Thus, in this embodiment, two dimensions of analysis, fluorescence channel and melting temperature, can be performed simultaneously in a single-tube reaction, that is, different targets can be detected by different signal collection temperatures using the same fluorescence channel, 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 signal collection temperature characteristics.

[0014] In some embodiments, the amplification reagent may further include a reagent that promotes a PCR reaction, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, etc. In some embodiments, the amplification reagent further includes, in addition to a polymerase having polymerization activity, other enzymes that act on nucleic acids, such as exonucleases, endonucleases, etc.

[0015] In some embodiments, two adjacent signal channel collections in the same signal channel refer to the signal collection temperatures used for the two signal channel collections being close to each other. For example, in some embodiments, in the same signal channel, if the reaction product is placed at a first temperature to perform a first signal channel collection, then the temperature is raised to a second temperature to perform a second signal channel collection, and then the temperature is raised to a third temperature to perform a third signal channel collection, the first signal channel collection and the second signal channel collection are two adjacent signal channel collections, and the second signal channel collection and the third signal channel collection are two adjacent signal channel collections, but the first signal channel collection and the third signal channel collection are not two adjacent signal channel collections.

[0016] In some embodiments, the signal collection temperatures used for two adjacent signal channel collections are made to differ by 4°C or more, thereby realizing that the difference in the number of types of target nucleic acids contained in the signals obtained by two adjacent signal channel collections in the same signal channel is one, thereby avoiding the situation where there is no difference in the target nucleic acid signals obtained by two adjacent signal channel collections, reducing the number of signal channel collections, and simplifying the operation steps.

[0017] In some embodiments, the signal collection temperature is 0-95°C. The selection of the signal collection temperature can be determined according to the actual situation of the primer probe composition designed by those skilled in the art. For example, according to the design of the primer probe composition, those skilled in the art can predict that the melting temperature of the reaction product representing the target nucleic acid is T, and when the signal of the target nucleic acid is to be detected, the signal collection temperature is ≦T, and when the signal of the target nucleic acid is not to be detected, the signal collection temperature is >T.

[0018] In some embodiments, each 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. In order to facilitate program design of the detection device, in some embodiments, when the primer probe composition has two types of probes, each signal collection is performed twice in two signal channels. In some embodiments, the test sample has five types of targets, and the primer probe composition for these five types of targets contains two types of probes, and 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. In this case, the signal collection is performed three times for 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, there are three types of corresponding targets, and the number of types of target nucleic acids contained in the signals obtained by the two signal collections at adjacent signal collection temperatures may differ by 0. In these embodiments, the number of signal collections is set based on the detection channel with the largest number of targets to be detected, so 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 made easier, and those skilled in the art can select according to actual needs. In some embodiments, those skilled in the art can reduce the number of signal collections and simplify the operation steps by choosing not to collect signals in channels that do not have desired targets according to the design situation of the primer probe composition.

[0019] In some embodiments, the method for detecting multiple target nucleic acids is a method for detecting multiple target nucleic acids by digital PCR. The method further comprises distributing the reaction system into 500 or more reaction cells before placing the reaction system under conditions that allow hybridization and extension reactions by nucleic acid polymerase, and each reaction cell contains one target nucleic acid of a test sample or does not contain a target nucleic acid of a test sample. In some embodiments, the signal collection comprises collecting a fluorescent signal by a camera. In some embodiments, the signal channel collection comprises collecting a fluorescent signal by a camera in a fluorescent signal channel.

[0020] In some embodiments, the primer probe composition, the test sample (including two types of targets) and the amplification reagent are mixed to obtain a reaction system, and then the reaction system is distributed into 500 or more reaction cells to form numerous droplets, each of which contains at most one target nucleic acid of the test sample, but also contains necessary components suitable for nucleic acid amplification, such as the primer probe composition and the amplification reagent, such as nucleic acid polymerase. Next, all the droplets are placed under conditions (in some embodiments, that is, PCR amplification conditions) that allow hybridization and extension reactions by nucleic acid polymerase, and the resulting reaction products (amplification products) are photographed a first time at a first temperature, some of the droplets emit light, and positive signals for the two targets are obtained, and then the temperature is raised to a second temperature and a second photograph is taken, and it is found that, among the droplets photographed a first time, some droplets do not emit light, and some droplets still emit light. Then, the droplets that still emit light in the second photograph are the first target, and the droplets that do not emit light in the second photograph but emit light in the first photograph are the second target. Based on the temperature and fluorescence channel used in the two images, the specific types of the two targets can be analyzed; by analyzing the number of luminescent droplets in the second image, the number of the first target can be obtained; and by analyzing the difference in the number of luminescent droplets in the first and second images, the number of the second target can be obtained.

[0021] In some embodiments, the conditions that allow hybridization and extension by the nucleic acid polymerase include pre-denaturation at about 85° C.-about 105° C. for 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 performing 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 performing a first PCR amplification on 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 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 performing 20-60 cycles.

[0022] In some embodiments, the conditions that allow hybridization and extension by the nucleic acid polymerase include pre-denaturation at 85° C.-105° C. for 0-15 minutes, denaturation at 85° C.-105° C. for 2-60 seconds, annealing and extension at 40° C.-75° C. for 10-90 seconds, and 20-60 cycles. In some embodiments, when the target nucleic acid in the test sample includes RNA, the amplification reagent further includes a reverse transcriptase, and the conditions that allow hybridization and extension by the nucleic acid polymerase include reverse transcription at 30° C.-65° C. for 2-30 minutes, pre-denaturation at 85° C.-105° C. for 0-15 minutes, denaturation at 85° C.-105° C. for 2-60 seconds, annealing and extension at 40° C.-75° C. for 10-90 seconds, and 20-60 cycles.

[0023] In some embodiments, in a method for detecting multiple types of target nucleic acids, the primer probe composition comprises a first probe and a first primer mixture, the first primer mixture comprises at least two types of primer sets, and different types of primer sets specifically bind to different types of target nucleic acids, respectively. 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 the first probe, and 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.

[0024] In some embodiments, the different types of primer sets in the first primer mixture generate different single-stranded pre-products with the corresponding target nucleic acids, and the different single-stranded pre-products generate different double-stranded products with the first probe, and the melting temperatures of the different double-stranded products are different. In some embodiments, the melting temperatures of the different double-stranded products differ by 4° C. or more.

[0025] The first probe is one type of probe that has one type of modified detection label that is the same 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).

[0026] "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, one type of primer set can also specifically bind to one type of non-separable other target nucleic acid, and this one type of non-separable target nucleic acid can also be considered as one type of target nucleic acid.

[0027] 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 certain target nucleic acid, which can specifically bind to its corresponding target nucleic acid and cannot specifically bind to other types of target nucleic acids.

[0028] 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, where one similar primer means that the primers have exactly the same sequence.

[0029] In this embodiment, for a primer set of a certain target nucleic acid, the generated first single-stranded pre-product is not extended after specifically binding with the probe (i.e., 0 bases are extended). 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, designing the generated first single-stranded pre-product to bind to the 5' end of the probe, or designing the generated first single-stranded pre-product to include a region at the 3' end that does not complementarily pair with the probe.

[0030] The melting temperature of an oligonucleotide is related to factors such as the length and composition of the oligonucleotide. Those skilled in the art can adjust the melting temperature of an oligonucleotide according to actual needs, for example, by increasing the GC content of the oligonucleotide and making the length of the oligonucleotide longer, and thus obtaining a higher melting temperature. Thus, those skilled in the art can adjust the sequence composition of the primer and / or probe according to actual circumstances to make the single-stranded pre-products formed by different primers different, and the positions at which the different single-stranded pre-products specifically bind to the first probe are different, and the melting temperatures of the different double-stranded products formed thereby are different. That is, those skilled in the art can make the double-stranded products representing different targets different according to actual circumstances, and the different double-stranded products have different melting temperatures, thereby obtaining signals of different target nucleic acids obtained at different signal collection temperatures. In some embodiments, the melting temperatures of different double-stranded products representing different targets can be made to differ by 4°C or more by designing the primer probe.

[0031] In some embodiments, the primer probe composition further comprises a second probe and a second primer mixture, the second probe having a different base sequence and a different modified detection label from 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.

[0032] 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 collect signals in different detection channels. In some embodiments, different detection labels do not mean that the modified detection groups are completely different. For example, if 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 some embodiments, the number of types of probes is increased, and 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.

[0033] In this embodiment, "first probe" and "second probe" are used to distinguish that the probe belongs 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.

[0034] In some embodiments, the primer sets in the second 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 second probe, the single-stranded pre-products form double-stranded products after specifically binding to the second probe and extending by ≧0 bases, and the formation of the double-stranded products causes a detectable signal change.

[0035] In some embodiments, the different types of primer sets in the second primer mixture generate different single-stranded pre-products with the corresponding target nucleic acids, and the different single-stranded pre-products generate different double-stranded products with the second probe, and the melting temperatures of the different double-stranded products are different. In some embodiments, the melting temperatures of the different double-stranded products differ by 4°C or more.

[0036] In some embodiments, in the primer probe composition, 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 the different probes are different from each other; the primer set includes a first primer and a second primer, the first primer includes a target sequence binding region 1, the second primer includes a primer signal detection region (h) and a target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2, the primer signal detection region (h) does not specifically bind to any target nucleic acid, and has a sequence that is partially or entirely the same 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 the different primer sets are different from each other.

[0037] 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.

[0038] In some embodiments, the primer signal detection region (h) having a sequence that is partially or completely the same as the probe signal detection region (H) of the probe means that it is a reverse complementary sequence (h') of the primer signal detection region (h) and can specifically bind to a partial or complete sequence of the probe signal detection region (H) of the probe.

[0039] In some embodiments, the first primer and the second primer specifically bind to the target nucleic acid, and then generate a pre-product comprising a 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.

[0040] In some embodiments, the sequences of the primer signal detection region (h) of the second primer in different primer sets are different from each other, that is, the bases and / or lengths of the sequences of the primer signal detection region (h) of the second primer in different primer sets are different, so that the sequences of the reverse complementary sequences (h') of different single-stranded pre-products are different, and different single-stranded pre-products form different double-stranded products after specifically binding with probes and extending 0 bases or >0 bases, and the melting temperatures of the different double-stranded products can be separated from each other; or different single-stranded pre-products form different double-stranded products after specifically binding with 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 temperature and / or detection channel to realize multiplex detection.

[0041] In some embodiments, there is a 0-20 base interval between the primer signal detection region (h) and the target sequence binding region 2 of the second primer.

[0042] 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 type of primer set of the first primer mixture or the second primer mixture further contains a probe anchor region (A), the probe anchor region (A) is located at the 5' end of the target sequence binding region 1, and the probe anchor region (A) does not specifically bind to any target nucleic acid but specifically binds to the primer anchor region (A').

[0043] The first and second primers in the above primer set specifically bind to a target nucleic acid to generate a pre-product, which includes a 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 are extended by ≧0 bases to form a double-stranded product, and the formation of the double-stranded product causes the probe to produce a detectable signal change.

[0044] 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 by the specific binding and after 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 by the specific binding of the single-stranded pre-product to the probe and after extension by ≧0 bases.

[0045] In some embodiments, the sequences of the target sequence binding region 1 of the first primers in different primer sets are different, and the sequences of the probe anchor region (A) of the first primers in different primer sets may be the same or different, preferably with a spacing of 0-20 bases between the probe anchor region (A) and the target sequence binding region 1 of the first primer.

[0046] In some embodiments of the present invention, the 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 the extension block region (M) and its complementary sequence do not specifically bind to any probe or any target nucleic acid.

[0047] The first primer and the second primer including the extension block region (M) each specifically bind to a target nucleic acid to generate a pre-product, 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 a probe and is extended 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), thereby causing the probe to produce a detectable signal change.

[0048] The terms "first primer mixture", "second primer mixture", etc. are used for explanatory purposes only to distinguish between 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".

[0049] 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 such 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 without extension after specifically binding to the probe has the lowest melting temperature.

[0050] In the present invention, the terms "first primer" and "second primer" are used for explanatory purposes only to distinguish between limited objects, and do not limit the order or hierarchical order in any way. 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".

[0051] 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, and 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 capable of causing a signal change by fluorescence resonance energy transfer with another first detection group.

[0052] In the present invention, the positions of the first and second detection groups in the probe allow the reverse complementary sequence (h') of the single-stranded pre-product to specifically bind to the probe (P) and elongate by ≧0 bases to form a double-stranded product, and if the positions of the first and second detection groups change due to the formation of the double-stranded product, the probe can be caused to produce a detectable signal change. The positions of the first and second detection groups are interchangeable.

[0053] 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 to be detected is present, the first primer and the second primer each specifically bind to the target sequence and extend to generate a double-stranded amplification product, of which the single-stranded amplification product and the probe specifically bind 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.

[0054] In some embodiments, the primer probe composition, the test sample and the amplification reagent are mixed to obtain a reaction system, and then the reaction system is placed under conditions that allow hybridization and extension reactions by a nucleic acid polymerase to obtain a reaction product.

[0055] PCR amplification is performed under conditions that allow hybridization and extension reactions by nucleic acid polymerase, and the following pre-products are obtained. The forward primer (F1) and reverse primer (R1) each specifically bind to target sequence 1 and extend to generate a double-stranded pre-amplification product, of which, from the 5' end to the 3' end of one single-stranded pre-amplification product (S1), 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') are obtained in this order. The forward primer (F2) and reverse primer (R2) each specifically bind to target sequence 2 and extend to generate a double-stranded pre-amplification product, of which, from the 5' end to the 3' end of one single-stranded pre-amplification product (S2), the probe anchor region (A2), the target sequence, and the reverse complementary sequence of the primer signal detection region (h2') are obtained in this order. Optionally, when a forward primer (F3) and a reverse primer (R3) are present, the forward primer (F3) and the reverse primer (R3) respectively specifically bind to the target sequence 3 and extend to generate a double-stranded pre-amplification product, of which, from the 5' end to the 3' end of one single-stranded pre-amplification product (S3), are the probe anchor region (A3), the target sequence, and the reverse complementary sequence (h3') of the primer signal detection region, in that order.

[0056] The pre-product is placed under conditions that allow hybridization and extension reaction by nucleic acid polymerase (constant temperature incubation, or PCR amplification conditions with several high and low temperature cycles) to obtain a reaction product. At this time, the probe signal detection region (H) of the probe (P) and the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded pre-amplification product (S1) are reverse complementary paired, the primer anchor region (A1') of the probe (P) is reverse complementary 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 with the probe (P) is obtained, the melting temperature of which is T1, and 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 (P) or the target sequence. At this time, the probe signal detection region (H) of the probe (P) 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), 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), and the 3' end of the single-stranded pre-amplification product (S2) continues to extend to the 5' end of the probe (P), thereby obtaining a partial or entire reverse complementary sequence of the probe (P); that is, the single-stranded pre-amplification product (S2) is used as a primer to complete secondary amplification with the probe (P) as a template, and an amplified double-stranded product (D2) formed with the probe (P) is obtained, the melting temperature of which is T2, where T2>T1.Optionally, when forward primer (F3) and reverse primer (R3) are present, the probe signal detection region (H) of probe (P) complementarily pairs with the reverse complementary sequence (h3') of the primer signal detection region at the 3' end of 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 single-stranded pre-amplification product (S3), and the 3' end of single-stranded pre-amplification product (S3) can be subsequently extended to the 5' end of probe (P) to obtain a part or all of the reverse complementary sequence of probe (P), that is, the single-stranded pre-amplification product (S3) is used as a primer and the probe (P) is used as a template to complete secondary amplification, and an amplified double-stranded product (D3) is formed with the probe (P), and its melting temperature is T3, where T3>T2.

[0057] In some embodiments, the reaction product is placed at n different signal collection temperatures to perform n signal collections, and then the presence or absence of a difference between the signals acquired in two signal collections at adjacent signal collection temperatures in the same signal channel is analyzed to determine whether the target nucleic acid is present in the test sample.

[0058] Perform the first signal acquisition at the first signal acquisition temperature (t1, t1 ≤ T1). Since the single-stranded amplification products S1, S2, and S3 respectively form double-stranded structures D1, D2, and D3, either completely or partially, with the probe (P), compared to the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes longer, the efficiency of fluorescence resonance energy transfer is low, the fluorescence signal changes, and it is detected by the device. Further, increase the temperature to the second signal acquisition temperature (t2, T1 < t2 ≤ T2) to perform the second signal acquisition. The amplified double-stranded product (D1) formed by the single-stranded pre-amplification 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 amplified double-stranded product (D1), and no fluorescence signal is generated. The amplified double-stranded products (D2) formed by the single-stranded pre-amplification product (S2) and the probe (P) and the amplified double-stranded products (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P) remain in the double-stranded state, and the fluorescence signal is detected by the device. Optionally, when the forward primer (F3) and the reverse primer (R3) are present, further increase the temperature to the third signal acquisition temperature (t3, T2 < t3 ≤ T3) to perform the third signal acquisition. The amplified double-stranded product (D2) formed by the single-stranded pre-amplification product (S2) and the probe (P) 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), and no fluorescence signal is generated. The amplified double-stranded product (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P) remains in the double-stranded state, and the fluorescence signal is detected by the device.

[0059] In some embodiments, place the reaction product at n different signal acquisition temperatures to perform n signal acquisitions. Then, in the same signal channel, analyze whether there is a difference in the presence or absence between the signals obtained from two adjacent signal acquisitions at the signal acquisition temperature to identify whether the target nucleic acid exists in the sample to be tested. Specifically, it includes the following content.

[0060] Optionally, when forward primer (F3) and reverse primer (R3) are present, the first signal collection is performed at the first signal collection temperature (t3, T2 < t3 ≤ T3). The amplified double-stranded product (D1) formed by the single-stranded pre-amplified product (S1) and the probe (P) and the amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P) cannot form a double-stranded structure because the first signal collection temperature (t3) is higher than the melting temperature of the amplified double-stranded products (D1 and D2). The amplified double-stranded product (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) has a longer distance between the first detection group and the second detection group compared to the state of the probe (P) before PCR, resulting in a lower fluorescence resonance energy transfer efficiency, a change in the fluorescence signal, and being detected by the device. Further, the temperature is decreased to the second signal collection temperature (t2, T1 < t2 ≤ T2) to perform the second signal collection. The amplified double-stranded product (D1) formed by the single-stranded pre-amplified product (S1) and the probe (P) cannot form a double-stranded structure because the second signal collection temperature (t2) is higher than the melting temperature of the amplified double-stranded product (D1), and no fluorescence signal is generated. The amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P) has a longer distance between the first detection group and the second detection group compared to the state of the probe (P) before PCR, resulting in a lower fluorescence resonance energy transfer efficiency, a change in the fluorescence signal, and being detected by the device. The amplified double-stranded product (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) remains in a double-stranded state and the fluorescence signal is detected by the device. Further, the temperature is decreased to the third signal collection temperature (t1, t1 ≤ T1) to perform the third signal collection. The amplified double-stranded product (D1) formed by the single-stranded pre-amplified product (S1) and the probe (P) has a longer distance between the first detection group and the second detection group compared to the state of the probe (P) before PCR, resulting in a lower fluorescence resonance energy transfer efficiency, a change in the fluorescence signal, and being detected by the device. The amplified double-stranded products (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) and the amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P) remain in a double-stranded state and the fluorescence signals are detected by the device.

[0061] According to a second aspect of the present invention, for accommodating a plurality of minute liquids, each of the minute liquids contains a reaction reagent, and some of the minute liquids further contain one of a first analyte or a second analyte, a reaction solution accommodating portion; a temperature adjusting portion for adjusting the temperature of the minute liquids in the reaction solution accommodating portion; a signal detecting portion for detecting a signal generated by the minute liquids in the reaction solution accommodating portion; controlling the temperature adjusting portion to adjust the temperature of the minute liquids in the reaction solution accommodating portion, and simultaneously generating signals in a plurality of minute liquids containing a first analyte or a second analyte; controlling the temperature adjusting portion to adjust the temperature of the minute liquids to t1, and a plurality of minute liquids containing the first analyte and a plurality of minute liquids containing the second analyte generate signals to form a first mixed signal; controlling the temperature adjusting portion to adjust the temperature of the minute liquids to t2, and only a plurality of minute liquids containing the second analyte generate signals to form a second signal; where t1 < t2, and a control portion that controls the signal detecting portion to collect signals generated by the minute liquids at temperatures t1 and t2 and output the first mixed signal and the second signal; a signal analyzing portion that calculates a first signal based on the first mixed signal and the second signal collected by the signal detecting portion, and an apparatus for detecting a plurality of types of target nucleic acids is provided.

[0062] In some embodiments, the control portion controls the temperature adjusting portion to adjust the temperature of the minute liquids in the reaction solution accommodating portion to t3, and a plurality of minute liquids containing the first analyte, a plurality of minute liquids containing the second analyte, and a plurality of minute liquids containing a third analyte generate signals to form a second mixed signal, where t3 < t1, and t3 and t1 differ by 4 °C or more.

[0063] In some embodiments, t1 and t2 differ by 4 °C or more.

[0064] In some embodiments, t1 and t2 differ by 4°C.

[0065] In some embodiments, the signal analysis unit subtracts the second signal from the first mixed signal to obtain the first signal.

[0066] In some embodiments, when the first mixed signal and the second signal are collected, the plurality of micro-fluids are spread out on the bottom of the reaction liquid storage section, and always maintain the same position state.

[0067] In some embodiments, the first signal is a fluorescent signal generated by a micro-fluid containing the first specimen, the second signal is a fluorescent signal generated by a micro-fluid containing the second specimen, and the first mixed signal is a fluorescent signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen.

[0068] In some embodiments, the signal analysis unit subtracts the fluorescent signal generated by a micro-fluid containing the second specimen at the same position from a first mixed signal generated by the micro-fluid to obtain the first signal.

[0069] In some embodiments, the signal analysis unit subtracts the first mixed signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen at the same position from the second mixed signal generated by the micro-fluid to obtain a third signal, which is a fluorescent signal generated by the micro-fluid containing the third specimen. In some embodiments, each of the micro-fluids includes a reaction reagent including an amplification substance for amplifying nucleic acid and a labeling substance for labeling nucleic acid, the amplification substance can amplify different nucleic acid molecules, and the labeling substance can bind to nucleic acid at a predetermined temperature to generate a detectable signal.

[0070] In some embodiments, the reaction reagents include a primer probe composition and amplification reagents, the amplification reagents including a nucleic acid polymerase and dNTPs, and the primer probe composition including a first probe and a first primer mix.

[0071] The first primer mixture contains at least two types of primer sets, and the different types of primer sets specifically bind to different types of target nucleic acids.

[0072] 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.

[0073] In some embodiments, different types of primer sets in the first primer mixture produce different single-stranded pre-products in combination with their corresponding target nucleic acids, and the different single-stranded pre-products produce different double-stranded products in combination with the first probe, and the melting temperatures of the different double-stranded products are different, and in some embodiments, the melting temperatures of the different double-stranded products differ by 4°C or more.

[0074] In some embodiments, the primer probe composition further comprises a second probe and a second primer mix.

[0075] The second probe and the first probe have different base sequences and are also differently modified detection labels, the second primer mixture contains at least one type of primer set, and the different types of primer sets specifically bind to different types of target nucleic acids.

[0076] In some embodiments, the primer sets in the second 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 second probe, the single-stranded pre-products form double-stranded products after specifically binding to the second probe and extending by ≧0 bases, and the formation of the double-stranded products causes a detectable signal change.

[0077] In some embodiments, different types of primer sets in the second primer mixture produce different single-stranded pre-products in combination with their corresponding target nucleic acids, and the different single-stranded pre-products produce different double-stranded products in combination with the second probe, and the melting temperatures of the different double-stranded products are different, preferably by 4°C or more.

[0078] In some embodiments, 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 region (H) of different probes are different from each other.

[0079] The primer set includes a first primer and a second primer, the first primer includes a target sequence binding region 1, and the second primer includes a primer signal detection region (h) and a target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2, the primer signal detection region (h) does not specifically bind to any target nucleic acid and has a sequence that is partially or entirely the same 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.

[0080] Preferably, the first probe or the second probe further comprises a primer anchor region (A'), and the first primer of at least one type of primer set of the first primer mixture or the second primer mixture further comprises a probe anchor region (A), the probe anchor region (A) is located at the 5' end of the target sequence binding region 1, and the probe anchor region (A) does not specifically bind to any target nucleic acid but specifically binds to the primer anchor region (A').

[0081] Preferably, 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), the extension block region (M) is located at the 5' end of the primer signal detection region (h), and the extension block region (M) and its complementary sequence do not specifically bind to any probe or any target nucleic acid. Effect of the Invention

[0082] Compared with the conventional techniques, the primer probe composition and digital PCR detection method of the present invention have the following advantages. (1) Simple method: The method described in the present invention can realize at least 8-plex reaction in digital PCR, and can distinguish different targets by distinguishing the presence or absence of fluorescence through a limited number of photographs, which has low requirements for fluorescence discrimination in digital PCR, is simple to operate, and saves time. (2) Multiplex detection. The method described in the present invention can simultaneously perform two-dimensional analysis of fluorescence channel and melting temperature in a single-tube reaction, that is, the same fluorescence channel can be used to detect different targets by different signal collection temperatures, or the detection of target types in different fluorescence channels can be realized as a product of the number of fluorescence channels and the signal collection temperature characteristics. (3) Low fluorescence background: By using only one type of probe for each fluorescence channel, the amplified products can be distinguished by their different melting temperatures, which greatly reduces the fluorescence background in PCR reactions and improves reaction sensitivity. (4) Melting temperature adjustable: In the method according to the present invention, the melting temperature of the secondary amplification product is adjusted by adjusting the length or sequence of the primer signal detection region (h) or adjusting the reverse complementary position between the primer signal detection region (h) and the entire signal detection region (H) of the probe (P) to increase or decrease the melting temperature of the secondary amplification double-stranded product formed with the probe (P), in order to distinguish the secondary amplification products by utilizing their different melting temperatures. (5) Good tolerance: The primer probe design method described in the present invention has a target sequence and two reverse complementary pairs, i.e., a forward primer (F) and a reverse primer (R), and compared with the Taqman hydrolysis probe method, which requires three parts to be reverse complementary to the template, when the target sequence has many hypermutation regions, such as virus or bacterial genomes, the primer probe design method described in the present invention has good tolerance and is less difficult to design. (6) Low cost: The number of fluorescent probes in a reagent can be reduced, significantly reducing reagent costs and enabling large-scale dissemination. (7) Single-tube reaction. The single-tube reaction reduces sample consumption and is particularly suitable for detecting rare samples. It can greatly increase the concentration of the sample added to the detection reaction and improve the detection sensitivity. The single-tube reaction also enables detection by the One-Tube method, which does not require subsequent extraction. (8) Simple operation: Only detection is performed using a digital PCR device, and there are no subsequent steps such as capillary electrophoresis or nucleic acid hybridization, and at the same time, the preparation is simple and no operation is required after input. (9) Less likely to be contaminated. The method described in the present invention is completely closed after sample addition is completed, and there is no need to open the lid for subsequent detection, which greatly reduces the possibility of contaminating the experimental environment. (10) 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. (11) 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. [Brief description of the drawings]

[0083] [Figure 1A] FIG. 1 is a signal diagram collected at 50° C. in Example 1. [Figure 1B] FIG. 1 is a signal diagram collected at 68° C. in Example 1. [Figure 2A] FIG. 13 is a signal diagram collected at 50° C. in Example 2. [Figure 2B] FIG. 13 is a signal diagram collected at 68° C. in Example 2. [Figure 2C] FIG. 13 is a signal diagram collected at 77° C. in Example 2. [Figure 3A] FIG. 11 is a signal diagram collected at 70° C. in Example 3. [Figure 3B] FIG. 11 is a signal diagram collected at 50° C. in Example 3. [Figure 4A] FIG. 13 is a signal diagram collected at 77° C. in Example 4. [Figure 4B] FIG. 11 is a signal diagram collected at 70° C. in Example 4. [Figure 4C] FIG. 11 is a signal diagram collected at 50° C. in Example 4. [Figure 5A] 1 is a schematic diagram of a detection device for multiple types of target nucleic acids disclosed in one embodiment of the present application. [Figure 5B] FIG. 2 is a schematic diagram of a detection device for multiple types of target nucleic acids disclosed in another embodiment of the present application. [Figure 5C] FIG. 13 is a structural diagram of a well plate comprising a plurality of reaction solution storage portions disclosed in another embodiment of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] 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.

[0085] In the following examples, detection and data analysis are performed using Maccura Biotechnology Co., Ltd.'s D600 fully automated digital PCR analysis system and reagent consumables. The digital PCR analysis system distributes a reaction system containing a sample, a primer probe composition and an amplification reagent into four reaction wells for amplification and detection (each reaction well contains multiple droplets, and each droplet is a reaction cell). In order to avoid redundant explanation, the drawings in the following examples provide droplet result diagrams of one of the reaction wells.

[0086] Example 1 (1) The dual detection primer probe system for detecting carbapenem antibiotic resistance gene VIM mutations and carbapenem antibiotic resistance gene KPC mutations is shown in Table 1.

[0087] [Table 1]

[0088] In the above primer probe, The forward primer F1-1 (SEQ ID NO:2) and the reverse primer R1-1 (SEQ ID NO:3) are both specific primers designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The total length of F1-1 is 39 bp, the 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P1 (SEQ ID NO:1). The total length of the R1-1 primer is 49 bp, the 1st to 25th bases at its 3' end are the target sequence binding region, the 6th to 21st bases at its 5' end are the same as the 16th to 31st bases at the 5' end of probe P1 (SEQ ID NO:1), and the 1st to 5th bases at its 5' end are the amplification blocking region. The forward primer F1-2 (SEQ ID NO: 4) and the reverse primer R1-2 (SEQ ID NO: 5) are both specific primers designed for the carbapenem antibiotic resistance gene KPC mutation target sequence. The total length of F2 is 37 bp, the 1st to 21st bases at its 3' end are a target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P1 (SEQ ID NO: 1). The total length of R1-2 primer is 35 bp, the 1st to 22nd bases at its 3' end are a target sequence binding region, and the 1st to 10th bases at its 5' end are completely identical to the 32nd to 41st bases at the 5' end of probe P1 (SEQ ID NO: 1).

[0089] (2) Using the above primer-probe system, two types of target nucleic acids were detected. The reaction system used for the detection is shown in Table 2.

[0090] [Table 2]

[0091] (3) The specific operating procedures upon detection are as follows: Sample preparation: A nucleic acid template of the carbapenem antibiotic resistance gene VIM mutation and a nucleic acid template of the carbapenem antibiotic resistance gene KPC mutation were used simultaneously to detect positive samples. Pure water was used as a templateless control (NTC). Reaction configuration: After sample preparation was completed, the reaction system was configured according to the mixing ratios shown in Table 2. Digital PCR amplification and signal collection: After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, then 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 (D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.). The amplification and collection process used was pre-denaturation at 95°C for 2 minutes, denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles, with the first signal collection at 50°C and the second signal collection at 68°C. Data analysis: The results were judged according to the positive droplets at different signal collection temperatures.

[0092] Figure 1A is a droplet diagram at a first signal collection temperature (50°C), where positive droplets (bright spots are positive droplets) can be seen to be distinguished from the background. Figure 1B is a droplet diagram at a second signal collection temperature (68°C), where positive droplets can be seen to be distinguished from the background, and the number of positive droplets has decreased compared to Figure 1A (some positive droplets in Figure 1A have no positive droplets at the corresponding positions in Figure 1B).

[0093] In Figure 1B, the positive droplets that are still present are signals from samples positive for the carbapenem antibiotic resistance gene KPC mutation, and the content of samples positive for the gene KPC mutation can be identified by analyzing the number of positive droplets. The positive droplets that are not present in Figure 1B but are present in Figure 1A are signals from samples positive for the carbapenem antibiotic resistance gene VIM mutation, and the content of samples positive for the gene VIM mutation can be identified by analyzing the difference between the positive droplets in Figure 1A and Figure 1B.

[0094] As can be seen from Figures 1A and 1B, the detection method of the present invention can realize the detection of multiple target nucleic acids. The realization of multiplex detection does not simply depend on the fluorescence channel, but uses different melting temperatures in the same fluorescence channel (i.e., the same signal channel) to distinguish the presence or absence of fluorescence through a limited number of photographs, so that different targets can be distinguished, which reduces the requirements for fluorescence discrimination in digital PCR, simplifies the operation, and saves time.

[0095] Example 2 (1) The triple primer probe system for detecting carbapenem antibiotic resistance gene VIM mutation, carbapenem antibiotic resistance gene KPC mutation, and carbapenem antibiotic resistance gene OXA-48 mutation is shown in Table 3.

[0096] [Table 3]

[0097] In the above primer probe, The forward primer F2-1 (SEQ ID NO:2) and the reverse primer R2-1 (SEQ ID NO:3) are both specific primers designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The total length of F2-1 is 39 bp, the 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P2 (SEQ ID NO:1). The total length of the R2-1 primer is 49 bp, the 1st to 25th bases at its 3' end are the target sequence binding region, the 6th to 21st bases at its 5' end are the same as the 16th to 31st bases at the 5' end of probe P2 (SEQ ID NO:1), and the 1st to 5th bases at its 5' end are the amplification blocking region. The forward primer F2-2 (SEQ ID NO: 4) and the reverse primer R2-2 (SEQ ID NO: 5) are both specific primers designed for the carbapenem antibiotic resistance gene KPC mutation target sequence. The total length of F2-2 is 37 bp, the 1st to 21st bases at its 3' end are a target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P2 (SEQ ID NO: 1). The total length of R2-2 primer is 35 bp, the 1st to 22nd bases at its 3' end are a target sequence binding region, and the 1st to 10th bases at its 5' end are completely identical to the 32nd to 41st bases at the 5' end of probe P2 (SEQ ID NO: 1). The forward primer F2-3 (SEQ ID NO: 6) and the reverse primer R2-3 (SEQ ID NO: 7) are both specific primers designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of F2-3 is 35 bp, the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P2 (SEQ ID NO: 1). The total length of R2-2 primer is 37 bp, 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 completely identical to the 1st to 10th bases at the 3' end of probe P2 (SEQ ID NO: 1).

[0098] (2) Three types of target nucleic acids were detected using the above primer-probe system. The reaction system used for the detection is shown in Table 4.

[0099] [Table 4]

[0100] (3) The specific operating procedures upon detection are as follows: Sample preparation: A nucleic acid template of the carbapenem antibiotic resistance gene VIM mutation, a nucleic acid template of the carbapenem antibiotic resistance gene KPC mutation, and a nucleic acid template of the carbapenem antibiotic resistance gene OXA-48 mutation were used simultaneously to detect positive samples. Pure water was used as a templateless control (NTC). Reaction configuration: After sample preparation was completed, the reaction system was configured according to the mixing ratios shown in Table 4. Digital PCR amplification and signal collection: After sealing the digital PCR tube with a lid, the sample was gently mixed uniformly, then 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 (D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.). The amplification and collection process used was pre-denaturation at 95°C for 2 minutes, denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles, constant temperature incubation at 40°C for 3 minutes, the first signal collection at 50°C, the second signal collection at 68°C, and the third signal collection at 77°C. Data analysis: The results were judged according to the positive droplets at different signal collection temperatures.

[0101] FIG. 2A is a droplet diagram at a first signal collection temperature (50° C.), where positive droplets (bright spots are positive droplets) can be seen to be distinguished from the background. FIG. 2B is a droplet diagram at a second signal collection temperature (68° C.), where positive droplets can be seen to be distinguished from the background, and the number of positive droplets has decreased compared to FIG. 2A (some positive droplets in FIG. 2A do not have positive droplets at the corresponding positions in FIG. 2B). FIG. 2C is a droplet diagram at a third signal collection temperature (77° C.), where positive droplets can be seen to be distinguished from the background, and the number of positive droplets has decreased compared to FIG. 2B (some positive droplets in FIG. 2B do not have positive droplets at the corresponding positions in FIG. 2C).

[0102] In FIG. 2C, the positive droplets still present are signals of carbapenem antibiotic resistance gene OXA-48 mutation positive samples, and the content of gene OXA-48 mutation positive samples can also be identified by analyzing the number of positive droplets. The positive droplets not present in FIG. 2C but present in FIG. 2B are signals of carbapenem antibiotic resistance gene KPC mutation samples, and the content of gene KPC mutation positive samples can also be identified by analyzing the difference between the positive droplets in FIG. 2B and FIG. 2C. The positive droplets not present in FIG. 2B but present in FIG. 2A are signals of carbapenem antibiotic resistance gene VIM mutation samples, and the content of gene VIM mutation positive samples can also be identified by analyzing the difference between the positive droplets in FIG. 2A and FIG. 2B.

[0103] As can be seen from Figures 2A-2C, the detection method of the present invention can realize the detection of multiple target nucleic acids. The realization of multiplex detection does not simply depend on the fluorescence channel, but uses different melting temperatures in the same fluorescence channel (i.e., the same signal channel) to distinguish the presence or absence of fluorescence through a limited number of photographs, so that different targets can be distinguished, which reduces the requirements for fluorescence discrimination in digital PCR, simplifies the operation, and saves time.

[0104] Example 3 (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 5.

[0105] [Table 5]

[0106] In the above primer probe, The forward primer F3-1 (SEQ ID NO: 9) and the reverse primer R3-1 (SEQ ID NO: 10) are both specific primers designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The total length of F3-1 is 39 bp, the 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P3 (SEQ ID NO: 8). The total length of the R3-1 primer is 49 bp, the 1st to 25th bases at its 3' end are the target sequence binding region, the 6th to 21st bases at its 5' end are the same as the 16th to 31st bases at the 5' end of probe P3 (SEQ ID NO: 8), and the 1st to 5th bases at its 5' end are the amplification blocking region. The forward primer F3-2 (SEQ ID NO: 11) and the reverse primer R3-2 (SEQ ID NO: 12) are both specific primers designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of F3-2 is 35 bp, the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P3 (SEQ ID NO: 8). The total length of R3-2 primer is 38 bp, the 1st to 24th bases at its 3' end are the target sequence binding region, and the 1st to 11th bases at its 5' end are completely identical to the 32nd to 42nd bases at the 5' end of probe P3 (SEQ ID NO: 8).

[0107] (2) Using the above primer-probe system, two types of target nucleic acids were detected. The reaction system used for the detection is shown in Table 6.

[0108] [Table 6]

[0109] (3) The specific operating procedures upon detection are as follows: Sample preparation: A nucleic acid template of the carbapenem antibiotic resistance gene VIM mutation and a nucleic acid template of the carbapenem antibiotic resistance gene OXA-48 mutation were used simultaneously to detect positive samples. Pure water was used as a templateless control (NTC). Reaction configuration: After sample preparation was completed, the reaction system was configured according to the mixing ratios shown in Table 6. Digital PCR amplification and signal collection: After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, then 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 (D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.). The amplification and collection process used was pre-denaturation at 95°C for 2 minutes, denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles, incubation at 52°C for 3 minutes, the first signal collection at 70°C, and the second signal collection at 50°C. Data analysis: The results were judged according to the positive droplets at different signal collection temperatures.

[0110] Figure 3A is a droplet diagram at a first signal collection temperature (70°C), where positive droplets (bright spots are positive droplets) can be seen to be distinguished from the background. Figure 3B is a droplet diagram at a second signal collection temperature (50°C), where positive droplets can be seen to be distinguished from the background, and the number of positive droplets has increased compared to Figure 3A (some negative droplets in Figure 3A have become positive droplets at the corresponding positions in Figure 3B).

[0111] The positive droplets present in Figure 3A are signals of samples positive for the carbapenem antibiotic resistance gene OXA-48 mutation, and the content of the samples positive for the gene OXA-48 mutation can be identified by analyzing the number of positive droplets. The positive droplets present in Figure 3B, but not present in Figure 3A, are signals of samples positive for the carbapenem antibiotic resistance gene VIM mutation, and the content of the samples positive for the gene VIM mutation can be identified by analyzing the difference between the positive droplets in Figure 3A and Figure 3B.

[0112] As can be seen from Figures 3A and 3B, the detection method of the present invention can realize the detection of multiple target nucleic acids. The realization of multiplex detection does not simply depend on the fluorescence channel, but uses different melting temperatures in the same fluorescence channel (i.e., the same signal channel) to distinguish the presence or absence of fluorescence through a limited number of photographs, so that different targets can be distinguished, which reduces the requirements for fluorescence discrimination in digital PCR, simplifies the operation, and saves time.

[0113] Example 4 (1) The triple primer probe system for detecting the carbapenem antibiotic resistance gene VIM mutation, the carbapenem antibiotic resistance gene OXA-48 mutation, and the carbapenem antibiotic resistance gene IMP mutation is shown in Table 7.

[0114] [Table 7]

[0115] The forward primer F4-1 (SEQ ID NO: 9) and the reverse primer R4-1 (SEQ ID NO: 10) are both specific primers designed for the carbapenem antibiotic resistance gene VIM mutation target sequence. The total length of F4-1 is 39 bp, the 1st to 23rd bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P4 (SEQ ID NO: 18). The total length of the R4-1 primer is 49 bp, the 1st to 25th bases at its 3' end are the target sequence binding region, the 6th to 21st bases at its 5' end are the same as the 16th to 31st bases at the 5' end of probe P4 (SEQ ID NO: 8), and the 1st to 5th bases at its 5' end are the amplification blocking region. The forward primer F4-2 (SEQ ID NO: 11) and the reverse primer R4-2 (SEQ ID NO: 12) are both specific primers designed for the carbapenem antibiotic resistance gene OXA-48 mutation target sequence. The total length of F4-2 is 35 bp, the 1st to 19th bases at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P4 (SEQ ID NO: 8). The total length of R4-2 primer is 38 bp, the 1st to 24th 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 32nd to 42nd bases at the 5' end of probe P4 (SEQ ID NO: 8). The forward primer F4-3 (SEQ ID NO: 13) and the reverse primer R4-3 (SEQ ID NO: 14) are both specific primers designed for the carbapenem antibiotic resistance gene IMP mutation target sequence. The total length of F4-3 is 40 bp, the 1st to 24th bases at its 3' end are a target sequence binding region, and the 1st to 13th bases at its 5' end are reverse complementary to the 1st to 13th bases at the 5' end of probe P4 (SEQ ID NO: 8). The total length of R4-3 primer is 35 bp, the 1st to 23rd bases at its 3' end are a target sequence binding region, and the 1st to 9th bases at its 5' end are the same as the 1st to 9th bases at the 3' end of probe P4 (SEQ ID NO: 8).

[0116] (2) Three types of target nucleic acids were detected using the above primer probe system. The reaction system used for detection is shown in Table 8.

[0117] [Table 8]

[0118] (3) The specific operating procedures upon detection are as follows: Sample preparation: A nucleic acid template of the carbapenem antibiotic resistance gene VIM mutation, a nucleic acid template of the carbapenem antibiotic resistance gene OXA-48 mutation, and a nucleic acid template of the carbapenem antibiotic resistance gene IPM mutation were used simultaneously to detect positive samples. Pure water was used as a templateless control (NTC). Reaction configuration: After sample preparation was completed, the reaction system was configured according to the mixing ratios shown in Table 8. Digital PCR amplification and signal collection: After sealing the digital PCR tube with a lid, the sample was gently mixed evenly, then 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 (D600 fully automated digital PCR analysis system from Maccura Biotechnology Co., Ltd.). The amplification and collection process used was pre-denaturation at 95°C for 2 minutes, denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles, constant temperature incubation at 40°C for 3 minutes, the first signal collection at 77°C, the second signal collection at 70°C, and the third signal collection at 50°C. Data analysis: The results were judged according to the positive droplets at different signal collection temperatures.

[0119] FIG. 4A is a droplet diagram at a first signal collection temperature (77° C.), where positive droplets (bright spots are positive droplets) can be seen to be distinguished from the background. FIG. 4B is a droplet diagram at a second signal collection temperature (70° C.), where positive droplets can be seen to be distinguished from the background, and the number of positive droplets has increased compared to FIG. 4A (some negative droplets in FIG. 4A have become positive droplets at the corresponding positions in FIG. 4B). FIG. 4C is a droplet diagram at a third signal collection temperature (50° C.), where positive droplets can be seen to be distinguished from the background, and the number of positive droplets has increased compared to FIG. 4B (some negative droplets in FIG. 4B have become positive droplets at the corresponding positions in FIG. 4C).

[0120] The positive droplets present in FIG. 4A are signals of carbapenem antibiotic resistance gene IMP mutation positive samples, and the content of gene IMP mutation positive samples can be identified by analyzing the number of positive droplets. The positive droplets not present in FIG. 4A but present in FIG. 4B are signals of carbapenem antibiotic resistance gene OXA-48 mutation samples, and the content of gene OXA-48 mutation positive samples can be identified by analyzing the difference between the positive droplets in FIG. 4A and FIG. 4B. The positive droplets not present in FIG. 4B but present in FIG. 4C are signals of carbapenem antibiotic resistance gene VIM mutation samples, and the content of gene VIM mutation positive samples can be identified by analyzing the difference between the positive droplets in FIG. 4B and FIG. 4C.

[0121] As can be seen from Figures 4A-4B, the detection method of the present invention can realize the detection of multiple target nucleic acids. The realization of multiplex detection does not simply depend on the fluorescence channel, but uses different melting temperatures in the same fluorescence channel (i.e., the same signal channel) to distinguish the presence or absence of fluorescence through a limited number of photographs, so that different targets can be distinguished, which reduces the requirements for fluorescence discrimination in digital PCR, simplifies the operation, and saves time.

[0122] EMBODIMENT 2 FOR CARRYING OUT THEINVENTION The embodiments of the present application provide a detection device for multiple types of target nucleic acids for multiplex detection of nucleic acids, and can distinguish different targets by distinguishing the presence or absence of fluorescence through a limited number of photographs, which places low requirements on fluorescence discrimination in digital PCR, is easy to operate, and saves time.

[0123] In order to allow those skilled in the art to better understand the solution of the present application, the technical solutions in the embodiments of the present application are described below with reference to the drawings in the embodiments of the present application, obviously, the described embodiments are only some embodiments of the present application, not all embodiments, and all the embodiments based on the present application should belong to the protection scope of the present application.

[0124] 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.

[0125] The terms "first", "second", etc., used in this application may be used to describe various elements herein, 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, a first specimen may be referred to as a second specimen, and similarly, a second specimen may be referred to as a first specimen, without departing from the scope of this application. Both the first specimen and the second specimen are specimens, but are not the same specimen. Furthermore, the term "multiple" used in the examples of this application means two or more than two.

[0126] The technical solutions of the present application are further described below in the form of examples.

[0127] The multiple target nucleic acid detection device of this embodiment is intended to detect a test sample, which may contain one type of test component, or two, three, four, five or more types of test components, and the nucleic acid detection device of this embodiment can detect multiple types of test components in the test sample.

[0128] 5A and 5B, the detection device for multiple types of target nucleic acids of this embodiment includes a reaction liquid storage section 1, a temperature control section 2, a signal detection section 3, a control section 4, and a signal analysis section 5. Here, the reaction liquid storage section 1 is for storing a test sample containing a test component and a reaction reagent capable of reacting with the test component, and provides a place for causing various reactions between the test sample and the reaction 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 reaction 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 controls 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 controls the signal detection section 3 to detect a signal due to a specific substance in the mixed liquid. Here, the signal analysis section 5 is for analyzing the signal collected by the signal detection section 3.

[0129] I. Test Sample The test sample is a body fluid from a human or animal body, and the test sample includes different test components, which may be nucleic acids or proteins. In this embodiment, the test component is nucleic acid to describe the technical solution. The test sample directly collected from the human or animal body can be subjected to a nucleic acid extraction and purification operation to extract nucleic acid, which may be performed manually or by a specialized nucleic acid extractor. The test sample directly collected from the human or animal body may not be subjected to a nucleic acid extraction and purification operation. The test sample includes different test nucleic acid molecules, such as a first test object (first test target nucleic acid), a second test object (second test target nucleic acid), a third test object (third test target nucleic acid), a fourth test object (fourth test target nucleic acid), a fifth test object (fifth test target nucleic acid), etc.

[0130] II. Reaction Reagents The reaction 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. In some embodiments, different test nucleic acids are labeled with the same labeling substance.

[0131] In some embodiments, the reaction reagent comprises a primer probe composition and an amplification reagent, the amplification reagent comprises a nucleic acid polymerase and dNTPs, and the primer probe composition comprises a probe and a primer mixture. The primer mixture comprises at least two types of primer sets, the different types of primer sets specifically bind to different types of target nucleic acids, and the primer sets in the 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, and 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. In some embodiments, the different types of primer sets in the first primer mixture generate different single-stranded pre-products with their corresponding target nucleic acids, and the different single-stranded pre-products generate different double-stranded products with the first probe, and the melting temperatures of the different double-stranded products are different. In some embodiments, the melting temperatures of the different double-stranded products differ by 4° C. or more.

[0132] 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 sufficient reaction reagents to amplify multiple target nucleic acids and label multiple target nucleic acids. For example, if there are two types of target nucleic acids to be tested in a test sample, i.e., a first test object and a second test object, after the micro-partitioning process, each first test object and each second test object are distributed into different microfluids, and there is a sufficient amount of reagent in one microfluid, which can not only meet the needs of amplification and labeling of the first test object in the microfluid, but also meet the needs of amplification and labeling of the second test object in the microfluid.

[0133] 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.

[0134] A container may be provided with a plurality of microcompartments, and the test sample and reaction reagent microfluids may be added to the plurality of microcompartments, so that each microcompartment contains at most one unit of nucleic acid molecule.

[0135] IV. Reaction solution storage section As shown in Figure 5A, 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.

[0136] As shown in FIG. 5B, the reaction solution 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.

[0137] In order to realize absolute quantitative detection of the test target nucleic acid, the microfluidic fluid containing the test nucleic acid and the reagent is enclosed in 500 or more, for example, about 20,000 microfluidic fluids, each of which contains one test nucleic acid molecule or no test nucleic acid molecule, and each of which is distributed into different microfluidic fluids regardless of how many types of test substances are present in the test sample (this is an ideal state, and a small amount of microfluidic fluid may contain one or more test target nucleic acid molecules).

[0138] 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.

[0139] As shown in FIG. 1C, the well plate 100 is composed of a plurality of reaction liquid storage sections 1, the bottom of which is flat, and a plurality of micro-liquids can be spread out on the bottom of the reaction liquid storage section 1.

[0140] V.Temperature control section As shown in FIG. 5A and FIG. 5B, 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. 5A, 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. 5B, 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.

[0141] 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.

[0142] 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. 5A) when it is necessary to detect a signal. As shown in FIG. 5B, the signal detection unit 3 may be fixedly provided relative to the reaction liquid storage unit.

[0143] 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 5A and 5B, the control unit 4 may be a single member capable of simultaneously controlling the temperature control unit and the signal detection unit.

[0144] VIII.Signal analysis section As shown in the figure, the signal analysis unit 5 is communicatively connected to the signal detection unit 3. The signal analysis unit performs analysis based on the signal collected by the signal detection unit, and acquires the signal of the test target nucleic acid, thereby acquiring information such as whether or not the test sample contains the test target nucleic acid, and the type and number of the test target nucleic acid contained.

[0145] Example 1 In this embodiment, a dual detection device and method are exemplified, in which a test sample includes a first test object (a first test target nucleic acid) and a second test object (a second test target nucleic acid). Note that a first test-related product (a test target nucleic acid undergoes operations such as polymerase chain reaction (PCR) and / or nucleic acid hybridization and / or nuclease cleavage to generate a corresponding test-related product, and the test-related product is usually a nucleic acid) generated from the first test object can bind to a labeling substance in a reagent. A second test-related product generated from a second test object can also bind to a labeling substance in a reagent. In this embodiment, in a certain temperature mode, the first test object generates a first test-related product and binds to a labeling substance, and at the same time, in the same temperature mode, the second test object generates a second test-related product and binds to a labeling substance. However, the separation temperatures of the two are different, 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 and second separation temperature ranges 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 t1, and the second test-related product can be separated from the labeled substance when the temperature is equal to or higher than t2, and the temperature t2 is higher than the temperature t1.

[0146] In this embodiment, in order to perform multiple detection for different test nucleic acids in a test sample and to distinguish between different targets (target nucleic acids) by distinguishing the presence or absence of fluorescence by taking images a limited number of times, a mixture of the test sample and reaction reagents (including a primer probe composition and an amplification reagent) is added to a reaction liquid storage section to form multiple micro-liquids, and the control section performs the following control.

[0147] The control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit, so that the target nucleic acid in the test sample generates a test-related product (the test nucleic acid includes a first test nucleic acid and a second test nucleic acid, but exists in different micro-fluids), during this process, the micro-fluid undergoes multiple temperature cycles, and in each temperature cycle, the micro-fluid changes to multiple different temperatures and continues at the temperature for a certain time, and the number of cycles may be multiple, for example, 20-60, for example, about 40. In each cycle, the micro-fluid reaches a high temperature state, and the double-stranded nucleic acid becomes a single-stranded nucleic acid, and then the micro-fluid reaches a low temperature state, and the single-stranded nucleic acid undergoes 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 number of different test target nucleic acids and / or different test related products in the test sample increases (i.e., the amount of test target nucleic acids and / or test related products in each microfluidic fluid increases, but the type of test nucleic acid remains the same), making them easier to detect. By appropriately setting the annealing temperature in the amplification process, the number of test nucleic acids and / or test related products increases, binds with the labeling substance in the reagent, and simultaneously generates signals in multiple microfluidic fluids containing the first test object or the second test object.

[0148] Before performing the 40 temperature cycles, the microfluidic solution may be first heated to a temperature t0 to pre-denaturate the target nucleic acid in the sample for a certain period of time (about 2-5 minutes). The pre-denaturation for a certain period of time is to convert the target nucleic acid from a double-stranded state to a single-stranded state.

[0149] After completing 40 cycles, the control unit controls the temperature adjustment unit to adjust the temperature of the microfluidic to t1, and the multiple microfluidic containing the first analyte and the multiple microfluidic containing the second analyte generate signals to form a first mixed signal. At temperature t1, the second analyte or the related product of the second analyte and the labeling substance are in a combined state to generate a signal, and the first analyte or the related product of the first analyte and the labeling substance are also in a combined state to generate a signal. Since a single test nucleic acid molecule is distributed into one microfluidic, that is, there is only one test nucleic acid molecule in one microfluidic, this test nucleic acid molecule may be the first test nucleic acid or may be the second test nucleic acid, and at temperature t1, both the microfluidic containing the first test nucleic acid and the microfluidic containing the second test nucleic acid can generate signals. When multiple microfluidic overlap, the signal emitted by such a microfluidic is an invalid signal, and the signal emitted by a single microfluidic that does not overlap is a valid signal.

[0150] Next, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid to t2 (t2>t1), and only the multiple micro-fluids containing the second analyte generate a signal to form a second signal. At temperature t2, the first analyte or the related product of the first analyte and the labeling substance are separated, so no detectable signal is generated, but the second analyte or the related product of the second analyte and the labeling substance are still in a combined state, so a signal is generated. Similarly, since a single nucleic acid molecule is distributed in one micro-fluid, i.e., there is only one nucleic acid molecule in one micro-fluid, this nucleic acid molecule may be the first analyte nucleic acid and may be the second analyte nucleic acid, and at temperature t2, only the multiple micro-fluids in which the second analyte nucleic acid is located can generate a signal. When multiple micro-fluids overlap, the signal emitted by such micro-fluids is an invalid signal, and the signal emitted by a single micro-fluid that does not overlap is a valid signal.

[0151] In order to better distinguish between the first mixed signal and the second signal, t1 and t2 differ by 4°C or more, for example, by 4-40°C, and also differ by, for example, 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C, or 4°C.

[0152] In order to obtain a more accurate first signal, when the first mixed signal and the second signal are collected, a plurality of the micro liquids are spread out on the bottom of the reaction liquid storage section and always maintained in the same position.

[0153] By the above-mentioned control by the control unit, when the signal detection unit detects a signal generated from the micro-fluid, it is possible to realize that the number of types of target nucleic acid contained in the signal obtained by collecting two adjacent signals differs by one type (i.e., the first mixed signal contains signals of the first and second test objects, while the second signal contains only the signal of the first test object).

[0154] In order to obtain the types and numbers of target nucleic acids in a test sample and to realize multiplex detection of different test nucleic acids in the test sample, the signal analysis unit performs the following operations.

[0155] A first signal is calculated based on the first mixed signal and the second signal collected by the signal detection unit. The signal analysis unit can obtain the first signal by subtracting the second signal from the first mixed signal. Specifically, the signal analysis unit obtains the first signal by subtracting the fluorescent signal generated by the micro-fluid containing the second specimen at the same position from the first mixed signal generated by the micro-fluid. Here, the first signal is a fluorescent signal generated by the micro-fluid containing the first specimen, the second signal is a fluorescent signal generated by the micro-fluid containing the second specimen, and the first mixed signal is a fluorescent signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen.

[0156] Based on the second signal collected by the signal detection unit, a fluorescent signal generated by the micro fluid containing the second specimen can be directly acquired.

[0157] Since a single test nucleic acid molecule is distributed in one microfluid, i.e., one microfluid contains at most one test nucleic acid molecule (if some microfluids do not contain test nucleic acid molecules, none of them will generate signals at each signal collection temperature), this test nucleic acid molecule may be the first test nucleic acid or may be the second test nucleic acid. The total number of the first and second test specimens is obtained by analyzing the number of microfluids that generate signals in the first mixed signal. The number of the second test specimens is obtained by analyzing the number of microfluids that generate signals in the second signal. The number of the first test specimens is obtained by subtracting the number of the second test specimens in the second signal from the total number of the first and second test specimens in the first mixed signal.

[0158] Example 2 In this embodiment, a triple detection device and method are exemplified in which a test sample includes a first analyte (first test target nucleic acid), a second analyte (second test target nucleic acid), and a third analyte (third test target nucleic acid). Similarly, a first test-related product generated by the first analyte (a test target nucleic acid generates a corresponding test-related product after undergoing operations such as polymerase chain reaction (PCR) and / or nucleic acid hybridization and / or nuclease cleavage, and the test-related product is usually a nucleic acid) can bind to a labeling substance in a reagent. A second test-related product generated by the second analyte can bind to a labeling substance in a reagent, and a third test-related product generated by the third analyte can bind to a labeling substance in a reagent. In this embodiment, in a certain temperature mode, the first analyte generates a first analyte-related product and binds to the labeling substance, and at the same time, in the same temperature mode, the second analyte generates a second analyte-related product and binds to the labeling substance, and the third analyte generates a third analyte-related product and binds to the labeling substance. However, the separation temperatures of the three are different, the first analyte-related product can be separated from the labeling substance in the reagent within the first separation temperature range, the second analyte-related product can be separated from the labeling substance in the reagent within the second separation temperature range, and the third analyte-related product can be separated from the labeling substance in the reagent within the third separation temperature range, and the minimum temperatures of the first separation temperature range, the second separation temperature range, and the third separation temperature range are different respectively. In this embodiment, the minimum temperature of the third separation temperature range is lower than the minimum temperature of the first separation temperature range, and the minimum temperature of the first separation temperature range is lower than the minimum temperature of the second 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 t1, the second test-related product can be separated from the labeled substance when the temperature is equal to or higher than t2, and the third test-related product can be separated from the labeled substance when the temperature is equal to or higher than t3, where <t1<t2である。

[0159] In this embodiment, in order to perform triple detection for different test nucleic acids in a test sample and to distinguish between different targets (target nucleic acids) by distinguishing the presence or absence of fluorescence by taking images a limited number of times, a mixture of the test sample and reaction reagents (including a primer probe composition and an amplification reagent) is added to a reaction liquid storage section to form multiple micro-liquids, and the control section performs the following control.

[0160] The control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit, so that the target nucleic acid in the test sample generates a test-related product (the target nucleic acid includes the first test object, the second test object and the third test object, but exists in different micro-fluids), during this process, the micro-fluid undergoes multiple temperature cycles, and in each temperature cycle, the micro-fluid changes to multiple different temperatures and continues at the temperature for a certain time, and the number of cycles may be multiple, for example, 20-60, for example, about 40. In each cycle, the micro-fluid reaches a high temperature state, and the double-stranded nucleic acid becomes a single-stranded nucleic acid, and then the micro-fluid reaches a low temperature state, and the single-stranded nucleic acid undergoes 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 number of different test target nucleic acids and / or different test related products in the test sample increases (i.e., the amount of test target nucleic acids and / or test related products in each microfluid increases, but the type of test nucleic acid remains the same), making them easier to detect. By appropriately setting the annealing temperature in the amplification process, the number of test nucleic acids and / or test related products increases, binds with the labeling substance in the reagent, and simultaneously generates signals in multiple microfluids containing the first test object, the second test object, or the third test object.

[0161] Before performing the 40 temperature cycles, the microfluidic solution may be first heated to a temperature t0 to pre-denaturate the target nucleic acid in the sample for a certain period of time (about 2-5 minutes). The pre-denaturation for a certain period of time is to convert the target nucleic acid from a double-stranded state to a single-stranded state.

[0162] After 40 cycles are completed, the control unit controls the temperature control unit to adjust the temperature of the micro-fluid to t3, and the multiple micro-fluids containing the first specimen, the multiple micro-fluids containing the second specimen, and the multiple micro-fluids containing the third specimen all generate signals to form a second mixed signal. At temperature t3, the second specimen or the related product of the second specimen and the labeling substance are in a combined state and generate signals, and the first specimen or the related product of the first specimen and the labeling substance are also in a combined state and generate signals at the same time, and the third specimen or the related product of the third specimen and the labeling substance are also in a combined state and generate signals. Since a single test nucleic acid molecule is distributed in one microfluidic fluid, i.e., there is only one test nucleic acid molecule in one microfluidic fluid, the test nucleic acid molecule may be the first test nucleic acid, may be the second test nucleic acid or may be the third test nucleic acid, and at temperature t3, the microfluidic fluid containing the first test nucleic acid, the microfluidic fluid containing the second test nucleic acid and the microfluidic fluid containing the third test nucleic acid can all generate signals. When multiple microfluidic fluids overlap, the signal emitted by such microfluidic fluids is an invalid signal, and the signal emitted by a single non-overlapping microfluidic fluid is a valid signal.

[0163] Next, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid to t1 (t1>t3), and the multiple micro-fluids containing the first analyte and the multiple micro-fluids containing the second analyte generate signals to form a first mixed signal. At temperature t1, the second analyte or the related product of the second analyte and the labeling substance are in a combined state and generate signals, and the first analyte or the related product of the first analyte and the labeling substance are also in a combined state and generate signals at the same time, but the third analyte or the related product of the third analyte and the labeling substance are also in a separated state and do not generate detectable signals. Since a single test nucleic acid molecule is distributed into one micro-fluid, that is, there is only one test nucleic acid molecule in one micro-fluid, this test nucleic acid molecule may be the first test nucleic acid or may be the second test nucleic acid. At temperature t1, both the micro-fluid containing the first test nucleic acid and the micro-fluid containing the second test nucleic acid can generate signals. When multiple microfluids overlap, the signal emitted by such microfluids is an invalid signal, whereas the signal emitted by a single non-overlapping microfluid is a valid signal.

[0164] Next, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid to t2 (t2>t1), and only the multiple micro-fluids containing the second analyte generate a signal to form a second signal. At temperature t2, the first analyte or the related product of the first analyte and the labeling substance are separated, and the third analyte or the related product of the third analyte and the labeling substance are separated, so that neither generates a detectable signal, but the second analyte or the related product of the second analyte and the labeling substance are still in a combined state, so that a signal is generated. Similarly, since a single nucleic acid molecule is distributed in one micro-fluid, i.e., there is only one nucleic acid molecule in one micro-fluid, at temperature t2, only the multiple micro-fluids in which the second analyte nucleic acid exists can generate a signal. When multiple micro-fluids overlap, the signal emitted by such micro-fluids is an invalid signal, and the signal emitted by a single micro-fluid that does not overlap is a valid signal.

[0165] In order to better distinguish the second mixed signal, the first mixed signal and the second signal, t1 and t2 differ by 4° C. or more, for example, 4-40° C., such as 4-30° C., 4-20° C., 4-18° C., 4-15° C., 4-12° C., 4-10° C., 4-8° C., 4-6° C., 4-5° C. or 4° C. t1 and t3 differ by 4° C. or more, for example, 4-40° C., such as 4-30° C., 4-20° C., 4-18° C., 4-15° C., 4-12° C., 4-10° C., 4-8° C., 4-6° C., 4-5° C. or 4° C.

[0166] In order to obtain more accurate first and third signals, when the second mixed signal, the first mixed signal and the second signal are collected, a plurality of the micro liquids are spread out on the bottom of the reaction liquid storage section and always maintained in the same position.

[0167] The above-mentioned control by the control unit makes it possible to realize that when the signal detection unit detects a signal generated from the micro-fluid, the number of types of target nucleic acid contained in the signal obtained by collecting two adjacent signals differs by one type (i.e., the second mixed signal contains signals of the first test object, the second test object and the third test object, the first mixed signal contains signals of the first test object and the second test object, but the second signal contains only the signal of the first test object).

[0168] In order to obtain the types and numbers of target nucleic acids in a test sample and to realize multiplex detection of different test nucleic acids in the test sample, the signal analysis unit performs the following operations.

[0169] A third signal is calculated based on the first mixed signal and the second mixed signal collected by the signal detection unit. The signal analysis unit can obtain the third signal by subtracting the first mixed signal from the second mixed signal. Specifically, the signal analysis unit obtains the third signal by subtracting the first mixed signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen at the same position from the second mixed signal generated by the micro-fluid. Here, the second mixed signal is a fluorescent signal generated by the micro-fluid containing the first specimen, the micro-fluid containing the second specimen, and the micro-fluid containing the third specimen, the first mixed signal is a fluorescent signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen, and the third signal is a fluorescent signal generated by the micro-fluid containing the third specimen.

[0170] A first signal is calculated based on the first mixed signal and the second signal collected by the signal detection unit. The signal analysis unit can obtain the first signal by subtracting the second signal from the first mixed signal. Specifically, the signal analysis unit obtains the first signal by subtracting the fluorescent signal generated by the micro-fluid containing the second specimen at the same position from the first mixed signal generated by the micro-fluid. Here, the first signal is a fluorescent signal generated by the micro-fluid containing the first specimen, the second signal is a fluorescent signal generated by the micro-fluid containing the second specimen, and the first mixed signal is a fluorescent signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen.

[0171] Based on the second signal collected by the signal detection unit, a fluorescent signal generated by the micro liquid containing the second specimen can be directly acquired.

[0172] Since a single test nucleic acid molecule is distributed in one microfluid, i.e., one microfluid contains at most one test nucleic acid molecule (if some microfluids do not contain a test nucleic acid molecule, none of them will generate a detectable signal at each signal collection temperature), the test nucleic acid molecule may be the first test nucleic acid, or may be the second test nucleic acid or the third test nucleic acid. The total number of the first test specimen, the second test specimen, and the third test specimen is obtained by analyzing the number of microfluids that generate a signal in the second mixed signal. The total number of the first test specimen and the second test specimen is obtained by analyzing the number of microfluids that generate a signal in the first mixed signal. The number of the second test specimen is obtained by analyzing the number of microfluids that generate a signal in the second signal. The number of the third test specimen is obtained by subtracting the total number of the first test specimen and the second test specimen in the first mixed signal from the total number of the first test specimen, the second test specimen, and the third test specimen in the second mixed signal. The number of first analytes is obtained by subtracting the number of second analytes in the second signal from the total number of the first analytes and the second analytes in the first mixed signal.

[0173] Example 3 In this embodiment, the test sample includes a fourth test object (fourth test target nucleic acid) and a fifth test object (fifth test target nucleic acid), and the device and method for dual detection are exemplified. Note that the fourth test-related product (the test target nucleic acid undergoes operations such as polymerase chain reaction (PCR) and / or nucleic acid hybridization and / or nuclease cleavage to generate a corresponding test-related product, and the test-related product is usually a nucleic acid) generated from the fourth test object can bind to a labeling substance in the reagent. Similarly, the fifth test-related product generated from the fifth test object can also bind to a labeling substance in the reagent. In this embodiment, in a certain temperature mode, the fourth test object generates a fourth test-related product and binds to a labeling substance, and at the same time, in the same temperature mode, the fifth test object generates a fifth test-related product and binds to a labeling substance. However, the separation temperatures of the two are different, the fourth test-related product can be separated from the labeled substance in the reagent within a fourth separation temperature range, and the fifth test-related product can be separated from the labeled substance in the reagent within a fifth separation temperature range, and the fourth separation temperature range and the minimum temperature of the fourth separation temperature range are different. In this embodiment, the minimum temperature of the fourth separation temperature range is higher than the minimum temperature of the fifth separation temperature range. For example, the fourth test-related product can be separated from the labeled substance when the temperature is equal to or higher than t4, and the fifth test-related product can be separated from the labeled substance when the temperature is equal to or higher than t5, and the temperature t4 is higher than the temperature t5.

[0174] In this embodiment, in order to perform multiple detection for different test nucleic acids in a test sample and to distinguish between different targets (target nucleic acids) by distinguishing the presence or absence of fluorescence by taking images a limited number of times, a mixture of the test sample and reaction reagents (including a primer probe composition and an amplification reagent) is added to a reaction liquid storage section to form multiple micro-liquids, and the control section performs the following control.

[0175] The control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluid in the reaction liquid storage unit, so that the target nucleic acid in the test sample produces a test-related product (the test nucleic acid includes the fourth test nucleic acid and the fifth test nucleic acid, but exists in different micro-fluids), during this process, the micro-fluid undergoes multiple temperature cycles, and in each temperature cycle, the micro-fluid changes to multiple different temperatures and continues at the temperature for a certain time, and the number of cycles can be multiple, for example, 20-60, for example, about 40. In each cycle, the micro-fluid reaches a high temperature state, and the double-stranded nucleic acid becomes a single-stranded nucleic acid, and then the micro-fluid reaches a low temperature state, and the single-stranded nucleic acid undergoes an annealing and elongation process (annealing and elongation can be performed at different temperatures, and the elongation temperature is higher than the annealing temperature). After multiple temperature cycles, the number of different test target nucleic acids and / or different test related products in the test sample will all increase (i.e., the amount of test target nucleic acids and / or test related products in each microfluidic fluid will all increase, but the type of test nucleic acid will remain the same), making them more likely to be detected. By appropriately setting the annealing temperature in the amplification process, the number of test nucleic acids and / or test related products will increase and bind with the labeling substance in the reagent, causing multiple microfluidic fluids containing the fourth or fifth test object to simultaneously generate signals.

[0176] Before performing the 40 temperature cycles, the microfluidic solution may be first heated to a temperature t0 to pre-denaturate the target nucleic acid in the sample for a certain period of time (about 2-5 minutes). The pre-denaturation for a certain period of time is to convert the target nucleic acid from a double-stranded state to a single-stranded state.

[0177] After 40 cycles are completed, the control unit controls the temperature adjustment unit to adjust the temperature of the microfluid to t4, and only the plurality of microfluids containing the fourth analyte generate signals to form a fourth signal. At temperature t4, since the fifth analyte or a related product of the fifth analyte and the labeling substance are separated, no detectable signal is generated, but the fourth analyte or a related product of the fourth analyte and the labeling substance are still in a bound state and a signal is generated. Similarly, since a single nucleic acid molecule is dispensed into one microfluid, that is, there is only one nucleic acid molecule in one microfluid, this nucleic acid molecule may be the fourth analyte nucleic acid or may be the fifth analyte nucleic acid. At temperature t4, only the plurality of microfluids containing the fourth analyte nucleic acid can generate signals. When a plurality of microfluids overlap, the signals emitted by such microfluids are invalid signals, and the signals emitted by non-overlapping single microfluids are valid signals.

[0178] Next, the control unit controls the temperature adjustment unit to adjust the temperature of the microfluid to t5 (t5 < t4), and the plurality of microfluids containing the fourth analyte and the plurality of microfluids containing the fifth analyte generate signals to form a third mixed signal. At temperature t5, the fourth analyte or a related product of the fourth analyte and the labeling substance are in a bound state and generate a signal, and also the fifth analyte or a related product of the fifth analyte and the labeling substance are in a bound state and generate a signal. Since a single analyte nucleic acid molecule is dispensed into one microfluid, that is, there is only one analyte nucleic acid molecule in one microfluid, this analyte nucleic acid molecule may be the fourth analyte nucleic acid or may be the fifth analyte nucleic acid. At temperature t5, the microfluids containing the fourth analyte nucleic acid and the microfluids containing the fifth analyte nucleic acid can both generate signals. When a plurality of microfluids overlap, the signals emitted by such microfluids are invalid signals, and the signals emitted by non-overlapping single microfluids are valid signals.

[0179] In order to better distinguish the fourth signal from the third mixed signal, t4 and t5 differ by more than 4°C, for example, by 4-40°C, and also differ by, for example, 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C, or 4°C.

[0180] In order to obtain a more accurate fifth signal, when the fourth signal and the third mixed signal are collected, a plurality of the micro liquids are spread out on the bottom of the reaction liquid storage section and always maintained in the same position.

[0181] By the above-mentioned control by the control unit, when the signal detection unit detects a signal generated from the micro-fluid, the number of types of target nucleic acid contained in the signals obtained by collecting two adjacent signals differs by one type (i.e., the fourth signal contains only the signal of the fourth test object, and the third mixed signal contains the signals of the fourth test object and the fifth test object).

[0182] In order to obtain the types and numbers of target nucleic acids in a test sample and to realize multiplex detection of different test nucleic acids in the test sample, the signal analysis unit performs the following operations.

[0183] A fifth signal is calculated based on the fourth signal and the third mixed signal collected by the signal detection unit. The signal analysis unit can obtain the fifth signal by subtracting the fourth signal from the third mixed signal. Specifically, the signal analysis unit obtains the fifth signal by subtracting the fluorescent signal generated by the microfluid containing the fourth specimen at the same position from the third mixed signal generated by the microfluid. Here, the fourth signal is a fluorescent signal generated by the microfluid containing the fourth specimen, the fifth signal is a fluorescent signal generated by the microfluid containing the fifth specimen, and the third mixed signal is a fluorescent signal generated by the microfluid containing the fourth specimen and the microfluid containing the fifth specimen.

[0184] Based on the fourth signal collected by the signal detection unit, a fluorescent signal generated by the micro liquid containing the fourth specimen can be directly acquired.

[0185] Since a single test nucleic acid molecule is distributed in one microfluid, i.e., one microfluid contains at most one test nucleic acid molecule (if some microfluids do not contain test nucleic acid molecules, none of them will generate signals at each signal collection temperature), this test nucleic acid molecule may be the fourth test nucleic acid, and may also be the fifth test nucleic acid. The number of microfluids that generate signals in the third mixed signal is analyzed to obtain the total number of the fourth test specimen and the fifth test specimen. The number of microfluids that generate signals in the fourth signal is analyzed to obtain the number of the fourth test specimen. The number of the fifth test specimen is obtained by subtracting the number of the fourth test specimen in the fourth signal from the total number of the fourth test specimen and the fifth test specimen in the third mixed signal.

[0186] Of course, based on the above examples, those skilled in the art can reasonably infer how to use the technical solutions disclosed in the present application to realize time-saving and efficient quadruplex nucleic acid detection (detecting four types of target nucleic acids in a test sample), quinplex nucleic acid detection (detecting five types of target nucleic acids in a test sample), sextuplex nucleic acid detection (detecting six types of target nucleic acids in a test sample) or more multiplexed nucleic acid detection, etc.

[0187] 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.

[0188] 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).

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] The above is a detailed description of the device and detection method for detecting multiple types of target nucleic acids disclosed in the examples of the present application. Although the principles and embodiments of the present application are described in this specification using specific examples, the above description of the examples is merely intended to help understand 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. mixing the primer probe composition, a test sample, and an amplification reagent containing a nucleic acid polymerase and dNTPs to obtain a reaction system; placing the reaction system under conditions that allow hybridization and extension reactions by a nucleic acid polymerase to obtain a reaction product; The reaction product is placed at n different signal collection temperatures to perform a total of n signal collections, one signal collection is performed at each of the signal collection temperatures, and each signal collection includes at least one signal channel collection; Analyzing whether there is a difference between signals obtained in two adjacent collections of the same signal channel to determine whether a target nucleic acid is present in the test sample; and / or Analyzing the presence or absence of a signal obtained in the same signal channel at the highest signal collection temperature employed, to determine whether or not the target nucleic acid is present in the test sample; A method for detecting multiple types of target nucleic acids, wherein n is an integer of ≧2 and n≦the number of types of target nucleic acids.

2. The method for detecting multiple types of target nucleic acids described in claim 1, characterized in that the number of types of target nucleic acids contained in the signals obtained in two adjacent signal channel collections in the same signal channel differs by at most one type, preferably the number of types of target nucleic acids contained in the signals obtained in two adjacent signal channel collections in the same signal channel differs by at most one type, preferably the signal obtained in the signal channel collection at the highest signal collection temperature contains at most one type of target nucleic acid.

3. 2. The method for detecting multiple types of target nucleic acids according to claim 1, wherein the signal collection temperatures used in two adjacent signal channel collections in the same signal channel differ by 4° C. or more.

4. The method for detecting multiple types of target nucleic acids described in claim 1, characterized in that each 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.

5. The method for detecting multiple types of target nucleic acids described in claim 1, further comprising distributing the reaction system into 500 or more reaction cells before placing the reaction system under conditions that allow hybridization and extension reactions by nucleic acid polymerase, each reaction cell containing one target nucleic acid of a test sample or no target nucleic acid of a test sample, 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.

6. The conditions for allowing hybridization and extension reaction by the nucleic acid polymerase include pre-denaturation at about 85°C to about 105°C for 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 performing 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 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 85°C to about 105°C for 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 performing 20 to 60 cycles.

7. the primer probe composition comprises a first probe and a first primer mixture; the first primer mixture includes at least two types of primer sets, each of which specifically binds to a different type of target nucleic acid; a primer set in the first primer mixture generates a pre-product after specifically binding to its corresponding target nucleic acid, the pre-product includes a single-stranded pre-product that specifically binds to a first probe, the single-stranded pre-product forms a double-stranded product after specifically binding to the first probe and extending by ≧0 bases, and the formation of the double-stranded product causes a detectable signal change; The method for detecting multiple types of target nucleic acids described in claim 1, characterized in that different types of primer sets in the first primer mixture preferably produce different single-stranded pre-products together with their corresponding target nucleic acids, and the different single-stranded pre-products produce different double-stranded products together with the first probe, and the melting temperatures of the different double-stranded products are different, preferably the melting temperatures of the different double-stranded products differ by 4°C or more.

8. The primer probe composition further comprises a second probe and a second primer mixture; the second probe has a different base sequence from the first probe and also has a different modified detection label; the second primer mixture includes at least one type of primer set, and the different types of primer sets specifically bind to different types of target nucleic acids; Preferably, the primer sets in the second 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 second probe, the single-stranded pre-products form double-stranded products after specifically binding to the second probe and extending by ≧0 bases, and the formation of the double-stranded products causes a detectable signal change; The method for detecting multiple types of target nucleic acids described in claim 6, characterized in that different types of primer sets in the second primer mixture preferably produce different single-stranded pre-products when combined with their corresponding target nucleic acids, and the different single-stranded pre-products form different double-stranded products when combined with the second probe, and the melting temperatures of the different double-stranded products are different, preferably the melting temperatures of the different double-stranded products differ by 4°C or more.

9. the first probe or the second probe has a sequence that does not specifically bind to any target nucleic acid, 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; the primer set comprises a first primer and a second primer, the first primer comprises a target sequence binding region 1, the second primer comprises a primer signal detection region (h) and a target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2, 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, Preferably, the first probe or the second probe further comprises a primer anchor region (A'), and the first primer of at least one type of primer set of the first primer mixture or the second primer mixture further comprises a probe anchor region (A), the probe anchor region (A) is located at the 5' end of the target sequence binding region 1, and the probe anchor region (A) does not specifically bind to any target nucleic acid but specifically binds to the primer anchor region (A'); Preferably, in the first primer mixture or the second primer mixture, the second primer in at most one type of primer set further comprises an extension block region (M) located at the 5' end of the primer signal detection region (h), and the extension block region (M) and its complementary sequence do not specifically bind to any probe or any target nucleic acid. The method for detecting multiple types of target nucleic acids described in claim 7 or 8, characterized in that

10. A reaction liquid storage section for storing a plurality of micro-liquids, each of the micro-liquids containing a reaction reagent, and a part of the micro-liquids further containing one of a first specimen or a second specimen; 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 adjust the temperature of the micro-liquid in the reaction liquid storage unit, and a signal is generated simultaneously in a plurality of micro-liquids including a first specimen or a second specimen; The temperature adjusting unit is controlled to adjust the temperature of the micro-liquid to t1, and the multiple micro-liquids containing the first specimen and the multiple micro-liquids containing the second specimen generate signals to form a first mixed signal; The temperature control unit is controlled to adjust the temperature of the micro-liquid to t2, and only the multiple micro-liquids containing the second test object generate signals to form a second signal; Here, t1<t2, and a control unit that controls the signal detection unit to collect signals generated by the micro-liquid at temperatures t1 and t2 and output the first mixed signal and the second signal; and a signal analysis unit that calculates a first signal based on the first mixed signal and a second signal collected by the signal detection unit.

11. The device for detecting multiple types of target nucleic acids described in claim 10, characterized in that the control unit controls the temperature adjustment unit to adjust the temperature of the micro-fluids in the reaction liquid storage unit to t3, and the multiple micro-fluids containing the first specimen, the multiple micro-fluids containing the second specimen, and the multiple micro-fluids containing the third specimen generate signals to form a second mixed signal, where t3<t1, and t3 and t1 differ by 4°C or more.

12. The device for detecting multiple types of target nucleic acids according to claim 10 or 11, wherein t1 and t2 differ by 4° C. or more.

13. The device for detecting multiple types of target nucleic acids according to claim 10 or 11, characterized in that t1 and t2 differ by 4°C.

14. 12. The apparatus for detecting multiple types of target nucleic acids according to claim 10 or 11, characterized in that the signal analysis unit subtracts the second signal from the first mixed signal to obtain the first signal.

15. An apparatus for detecting multiple types of target nucleic acids as described in claim 10 or 11, characterized in that when collecting the first mixed signal and the second signal, the multiple micro-fluids are spread out on the bottom of the reaction liquid storage section and always maintain the same positional state.

16. The device for detecting multiple types of target nucleic acids described in claim 14 or 15, characterized in that the first signal is a fluorescent signal generated by a micro-fluid containing the first specimen, the second signal is a fluorescent signal generated by a micro-fluid containing the second specimen, and the first mixed signal is a fluorescent signal generated by a micro-fluid containing the first specimen and a micro-fluid containing the second specimen.

17. The device for detecting multiple types of target nucleic acids described in claim 16, characterized in that the signal analysis unit obtains the first signal by subtracting the fluorescent signal generated by a micro-fluid containing the second test substance at the same position from the first mixed signal generated by the micro-fluid.

18. The device for detecting multiple types of target nucleic acids described in claim 11, characterized in that the signal analysis unit subtracts the first mixed signal generated by the micro-fluid containing the first specimen and the micro-fluid containing the second specimen at the same position from the second mixed signal generated by the micro-fluid to obtain a third signal, which is a fluorescent signal generated by the micro-fluid containing the third specimen.

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