Multiplexed nucleic acid detection method using a probe-mediated signal system

Abstract

The present application relates to a nucleic acid detection method, specifically, the method of the present application introduces a nucleic acid complex, the nucleic acid complex is specifically combined with target nucleic acid, under the action of DNA polymerase, the nucleic acid complex is hydrolyzed, so that the melting curve is greatly reduced, so that the detection result is obtained.The method of the present application can detect multiple target nucleic acids simultaneously, has good specificity, simple operation, low cost, rapidness and wide application scene.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to a method for multiplex nucleic acid detection using a probe-mediated signaling system. Background Technology

[0002] In various pathogen detection scenarios, such as HPV infection, respiratory tract infection, intestinal infection, and bloodstream infection, there is a wide variety of pathogens requiring rapid detection. Therefore, there is an urgent need for rapid, multiplex, and low-cost nucleic acid detection technologies. Existing multiplex nucleic acid detection technologies mainly include: multiplex real-time quantitative PCR, reverse dot blot hybridization, and flow cytometry.

[0003] Real-time PCR technology offers unparalleled advantages over previous endpoint-based PCR techniques. Firstly, it is not only simple, rapid, and efficient, but also possesses high sensitivity and specificity. Secondly, because amplification and real-time measurement are performed in a closed system, the possibility of contamination is greatly reduced, and post-amplification manipulation is unnecessary. Furthermore, it allows for the simultaneous amplification of multiple target genes in the same reaction system using different primer designs—a process known as multiplex amplification. Quantitative PCR utilizes various probes, including TaqMan probes, molecular beacons, and scorpions, which offer high analytical sensitivity. However, due to current technological limitations, most instruments only offer four fluorescent dyes, thus limiting the detection of a maximum of four different targets per tube. Currently, some quantitative PCR technologies, by improving the dye system and the instrument's ability to interpret different wavelengths, can increase the detection capacity to ten targets per tube. However, the cost of these new dyes and instruments far exceeds that of conventional dyes and instruments.

[0004] Reverse dot blot hybridization is a target sequence fragment amplification based on PCR. The PCR method uses type-specific or universal primers to amplify the target fragment and hybridize it with specific probes immobilized on nitrocellulose membranes and solid-state chips. The presence of DNA template in the PCR system is determined by the presence of PCR products that hybridize with the specific probes immobilized on the nitrocellulose membranes and solid-state chips. However, reverse dot blot hybridization is difficult to automate, and the open detection environment is highly susceptible to cross-contamination, requiring extremely high levels of operator skill and a suitable laboratory environment.

[0005] Flow cytometry can detect up to 99 different targets by adding different proportions of fluorescent dyes to microspheres. As a result, commercial kits are available. However, the detection method is based on the detection of PCR products. The signal value reflects the concentration of the final PCR product and is not highly correlated with the concentration of the original virus. In addition, since the PCR product and probe need to be single-stranded when hybridizing, the high temperature process required for melting is not conducive to the application of automation technology.

[0006] For the need for multiplex detection, in addition to the technologies mentioned above, there are many solutions based on fluorescence PCR. For example, patent CN 105087827 uses 8 reaction tubes, each labeled with a different color fluorescent group, to detect 16 types of HPV. However, an obvious problem with this method is the large number of reaction tubes and PCR reaction materials used. Furthermore, a 96-well fluorescence PCR instrument can only detect 12 samples at a time, which is not ideal in terms of detection speed and cost. In patent CN 106048081, primers are used in conjunction with SYBR Green dye for HPV genotyping. The detection speed and cost are controlled. However, because only one dye is used, it is necessary to place the sample at 70-90℃ for 15 different melting peaks. This means that the Tm values ​​of these different melting peaks differ by only 1℃, which places extremely high demands on reagent stability and instrument. The problem of false positives caused by the melting peak temperatures being too close is a significant drawback.

[0007] Therefore, there is an urgent need in this field to develop a rapid, sensitive, highly specific, and accurate multiplex nucleic acid amplification and detection method based on existing dye labeling methods and instrument performance. Summary of the Invention

[0008] The purpose of this invention is to provide a probe-based multiplex nucleic acid detection method.

[0009] A first aspect of the present invention provides a nucleic acid detection method, the method comprising the steps of:

[0010] (s1) Provides a premix, a sample to be tested, and a DNA polymerase, wherein the premix contains a nucleic acid complex formed by a first nucleic acid, a second nucleic acid, and a third nucleic acid through complementary base pairing;

[0011] (s2) The test sample is mixed with the premix and DNA polymerase, so that the target nucleic acid in the test sample and the second nucleic acid base in the nucleic acid complex are complementary and pair to form a double-stranded structure.

[0012] PCR reaction is carried out under the action of DNA polymerase, which causes the second nucleic acid to be cleaved and extended using the first nucleic acid as a template, thereby hydrolyzing the third nucleic acid that is complementary to the first nucleic acid;

[0013] (s3) Perform melting curve analysis to obtain nucleic acid detection results;

[0014] The first nucleic acid has the structure shown in formula (I) from 5' to 3':

[0015] B1-B2(I)

[0016] In the formula, B1 consists of 10 to 30 nucleotides;

[0017] B2 consists of 15–30 nucleotides;

[0018] The second nucleic acid has the structure shown in formula (II) from 5' to 3':

[0019] J1-J2(II)

[0020] In the formula, J1 and B2 are complementary;

[0021] J2 is complementary to the target nucleic acid;

[0022] The third nucleic acid is complementary to B1.

[0023] In another preferred embodiment, the third nucleic acid is modified with a fluorescent group and a quenching group.

[0024] In another preferred embodiment, the second nucleic acid is phosphorylated at its 3' end.

[0025] In another preferred embodiment, the fluorescent group is selected from the group consisting of FAM, VIC, ROX, CY5, or combinations thereof.

[0026] In another preferred embodiment, the quenching group is selected from the group consisting of BHQ, TAMRA, DABCYL, or combinations thereof.

[0027] In another preferred embodiment, B1 is 15 to 30 nucleotides, more preferably 15 to 25 nucleotides, and even more preferably 15 to 23 nucleotides.

[0028] In another preferred embodiment, B2 is 15 to 25 nucleotides, more preferably 15 to 23 nucleotides, and even more preferably 16 to 23 nucleotides.

[0029] In another preferred embodiment, the length of the first nucleic acid is 30 to 50 nucleotides, more preferably 30 to 45 nucleotides, and even more preferably 30 to 40 nucleotides.

[0030] In another preferred embodiment, the second nucleic acid is 30 to 50 nucleotides in length, more preferably 30 to 45 nucleotides, and even more preferably 30 to 40 nucleotides.

[0031] In another preferred embodiment, the length of the third nucleic acid is 10 to 30 nucleotides, more preferably 15 to 25 nucleotides, and even more preferably 15 to 20 nucleotides.

[0032] In another preferred embodiment, the Tm of the first nucleic acid and the second nucleic acid are each independently 20-70℃, preferably 30-70℃.

[0033] In another preferred embodiment, when the method detects multiple targets, the third nucleic acid corresponding to different targets is modified with the same or different fluorescent groups.

[0034] In another preferred embodiment, when the method detects multiple targets, the melting temperatures of the third nucleic acid corresponding to different targets and modified with the same fluorescent group are different.

[0035] In another preferred embodiment, the melting curve Tm value interval between the third nucleic acids corresponding to different targets is ≥5℃, preferably ≥6℃, ≥7℃, ≥8℃, or ≥9℃, more preferably ≥10℃.

[0036] In another preferred embodiment, the first nucleic acid has a nucleotide sequence as shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

[0037] In another preferred embodiment, the second nucleic acid has a nucleotide sequence as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:26.

[0038] In another preferred embodiment, the third nucleic acid has a nucleotide sequence as shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0039] In another preferred embodiment, in step (s1), the molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1-10:1-5:1-10, more preferably 1-8:1-3:1-8, more preferably 1-5:1-3:1-5, for example, 2:1:2.

[0040] In another preferred embodiment, in step (s1), the concentration of the first nucleic acid in the premixed solution is 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0041] In another preferred embodiment, in step (s1), the concentration of the second nucleic acid in the premixed solution is 1 nM to 300 mM, more preferably 10 nM to 50 mM, more preferably 20 to 500 nM, for example about 50 nM.

[0042] In another preferred embodiment, in step (s1), the concentration of the third nucleic acid in the premixed solution is 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0043] In another preferred embodiment, in step (s2), the amount of DNA polymerase is 0.5 to 5 U, more preferably 0.5 to 3 U, more preferably 0.5 to 2 U, for example, about 1 U.

[0044] In another preferred embodiment, in step (s2), the DNA polymerase operates in a mixture containing a selection from the group consisting of: 10×PCR buffer, 0.05–1 mM dNTP, and 1–5 mM MgCl2.

[0045] In another preferred embodiment, in step (s2), the reaction program of the PCR reaction is 95℃ for 10 min; 95℃ for 15 s, 60℃ for 20 s, 72℃ for 15 s, repeated 40 to 50 times.

[0046] In another preferred embodiment, the premix further includes upstream and downstream primers for amplifying the target nucleic acid.

[0047] In another preferred embodiment, the upstream primer has a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, or SEQ ID NO:24.

[0048] In another preferred embodiment, the downstream primer has a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, or SEQ ID NO:25.

[0049] In another preferred embodiment, the concentrations of the upstream and downstream primers are each independently 50–500 nM, more preferably 100–400 nM, more preferably 150–300 nM, for example, about 200 nM.

[0050] In another preferred embodiment, the amount of the sample to be tested is 1 to 100 μL, more preferably 1 to 50 μL, more preferably 1 to 10 μL, for example, about 5 μL.

[0051] In another preferred embodiment, the sample to be tested is subjected to reverse transcription.

[0052] In another preferred embodiment, step (s3) includes heating at 95°C for 3 minutes, cooling to 20°C, heating to 70°C at a rate of 0.06°C / s, and detecting the fluorescence signal to obtain the detection result.

[0053] In another preferred embodiment, the method includes the steps of: providing a negative standard and performing melting curve analysis on the negative standard to obtain the melting curve height H0 of the negative standard.

[0054] In another preferred embodiment, the criterion for the method is that, compared with the melting curve height H0 of the negative standard, the melting curve height H1 of the test sample decreases by more than 10%, that is, (H0-H1) / H0>10%, indicating that the test sample contains target nucleic acid.

[0055] In another preferred embodiment, the method includes the steps of: providing a positive standard and performing melting curve analysis on the positive standard to obtain the melting curve height H2 of the positive standard.

[0056] In another preferred embodiment, if the melting curve height H2 of the positive standard decreases by more than 50% compared to the melting curve height H0 of the negative standard, i.e. (H0-H2) / H0>50%, it indicates that the target nucleic acid is present in the positive standard, and the method is effective; if the melting curve height H2 of the positive standard does not decrease by more than 50%, it indicates that the method is ineffective and retesting is required (quality control method).

[0057] In another preferred embodiment, the sample to be tested contains nucleic acids selected from the group consisting of influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human papillomavirus, or combinations thereof.

[0058] In another preferred embodiment, the detection limit of the method is ≤100 copies, more preferably ≤50 copies, and even more preferably ≤25 copies.

[0059] In another preferred embodiment, the method is a diagnostic method.

[0060] In another preferred embodiment, the method is for non-disease diagnosis purposes.

[0061] In a second aspect, the present invention provides a nucleic acid detection system, the nucleic acid detection system comprising:

[0062] (a) The first nucleic acid, which has the structure shown in formula (I) from 5' to 3':

[0063] B1-B2(I)

[0064] In the formula, B1 consists of 10 to 30 nucleotides;

[0065] B2 consists of 15–30 nucleotides;

[0066] (b) A second nucleic acid, which has the structure shown in formula (II) from 5' to 3':

[0067] J1-J2(II)

[0068] In the formula, J1 and B2 are complementary;

[0069] J2 is complementary to the target nucleic acid; and

[0070] (c) A third nucleic acid, which is complementary to B1.

[0071] In another preferred embodiment, in the nucleic acid detection system, the first nucleic acid, the second nucleic acid, and the third nucleic acid form a nucleic acid complex through complementary base pairing.

[0072] In another preferred embodiment, the first nucleic acid, the second nucleic acid, and the third nucleic acid are defined as described in the first aspect of the present invention.

[0073] In another preferred embodiment, the nucleic acid detection system further comprises DNA polymerase and / or PCR reaction solution.

[0074] In another preferred embodiment, the PCR reaction solution comprises 10×PCR buffer, 0.05–1 mM dNTP, and 1–5 mM MgCl2.

[0075] In another preferred embodiment, the nucleic acid detection system further includes primers for amplifying the target nucleic acid.

[0076] In another preferred embodiment, the primers include an upstream primer and a downstream primer, the upstream primer having a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, or SEQ ID NO:24; and the downstream primer having a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, or SEQ ID NO:25.

[0077] In another preferred embodiment, the concentrations of the upstream and downstream primers in the nucleic acid detection system are each independently 50–500 nM, more preferably 100–400 nM, more preferably 150–300 nM, for example, about 200 nM.

[0078] In another preferred embodiment, the first nucleic acid has a nucleotide sequence as shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

[0079] In another preferred embodiment, the second nucleic acid has a nucleotide sequence as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, or SEQ ID NO:26.

[0080] In another preferred embodiment, the third nucleic acid has a nucleotide sequence as shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0081] In another preferred embodiment, in the nucleic acid detection system, the molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1–10:1–5:1–10, more preferably 1–8:1–3:1–8, more preferably 1–5:1–3:1–5, for example, 2:1:2.

[0082] In another preferred embodiment, the concentration of the first nucleic acid in the nucleic acid detection system is 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0083] In another preferred embodiment, the concentration of the second nucleic acid in the nucleic acid detection system is 1 nM to 300 mM, more preferably 10 nM to 50 mM, and even more preferably 20 to 500 nM, for example, about 50 nM.

[0084] In another preferred embodiment, the concentration of the third nucleic acid in the nucleic acid detection system is 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0085] In another preferred embodiment, the amount of DNA polymerase in the nucleic acid detection system is 0.5–5 U, more preferably 0.5–3 U, more preferably 0.5–2 U, for example, about 1 U.

[0086] In another preferred embodiment, the nucleic acid detection system further includes a mixture selected from the group consisting of: 10×PCR buffer, 0.05–1 mM dNTP, and 1–5 mM MgCl2.

[0087] In a third aspect, the present invention provides a nucleic acid detection kit, the kit comprising:

[0088] (i) A first container and a nucleic acid detection system as described in the second aspect of the invention located within the first container.

[0089] In another preferred embodiment, the kit further comprises:

[0090] (ii) The second container and the DNA polymerase located within the second container;

[0091] (iii) the third container and the PCR reaction solution located within the third container; and / or

[0092] (iv) The fourth container and the primers located within the fourth container.

[0093] In another preferred embodiment, the first container, the second container, the third container, and / or the fourth container are the same or different containers.

[0094] In a fourth aspect, the present invention provides a nucleic acid detection method, comprising the steps of:

[0095] After performing nucleic acid amplification reaction on the sample to be tested using the nucleic acid detection system as described in the second aspect of the present invention or the detection kit as described in the third aspect of the present invention, melting curve analysis is performed to obtain the detection results.

[0096] In a fifth aspect, the present invention provides the use of a nucleic acid detection system as described in the second aspect of the present invention or a detection kit as described in the third aspect of the present invention for detecting nucleic acids in a sample to be tested.

[0097] In another preferred embodiment, the sample to be tested contains pathogens.

[0098] In another preferred embodiment, the pathogen includes a virus, preferably human papillomavirus, influenza virus, respiratory syncytial virus, and / or adenovirus.

[0099] In another preferred embodiment, the use is for disease diagnosis purposes.

[0100] In another preferred embodiment, the use is for non-disease diagnosis purposes.

[0101] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0102] Figure 1 A schematic diagram of the structure of the detection probe, signal probe, and tag sequence is shown.

[0103] Figure 2 A schematic diagram of this invention is shown.

[0104] Figure 3 The results of HPV detection using the method of the present invention are shown.

[0105] Figure 4 The results of the method of the present invention for simultaneously detecting influenza virus, respiratory syncytial virus and adenovirus are shown. Detailed Implementation

[0106] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a nucleic acid detection method based on nucleic acid complexes. This method involves the specific binding of the nucleic acid complex to the target nucleic acid, followed by hydrolysis of the nucleic acid complex by DNA polymerase, resulting in a decrease in the melting curve height and thus obtaining the detection result. This method can simultaneously detect multiple target nucleic acids, exhibits high specificity, is simple to operate, low in cost, rapid, and has a wide range of applications. This invention was completed based on this discovery.

[0107] the term

[0108] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0109] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0110] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0111] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0112] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0113] As used in this article, the terms "first nucleic acid" and "tag sequence" are used interchangeably.

[0114] As used in this article, the terms "second nucleic acid" and "detection probe" are used interchangeably.

[0115] As used in this article, the terms "third nucleic acid" and "signal probe" are used interchangeably.

[0116] As used herein, the term "nucleic acid complex" refers to a structure that is both single-stranded and double-stranded, formed by a tag sequence, a detection probe, and a signal probe through base complementarity pairing. Specifically, the signal probe is complementary to a portion of the tag sequence, a portion of the detection probe is complementary to another portion of the tag sequence, and another portion of the detection probe is complementary to the target nucleic acid sequence.

[0117] As used in this article, the term "Rm" refers to the fluorescence value variable corresponding to different melting peak values. This fluorescence value variable is automatically output by the device and is obtained by subtracting the background of the sample (automatically removed by the device) from the absolute value of the melting peak value displayed in the melting curve spectrum.

[0118] The detection method of the present invention

[0119] The detection method of the present invention includes the following steps:

[0120] (s1) Provides a premix, a sample to be tested, and a DNA polymerase, wherein the premix contains a nucleic acid complex formed by a first nucleic acid, a second nucleic acid, and a third nucleic acid through complementary base pairing;

[0121] (s2) The test sample is mixed with the premix and DNA polymerase, so that the target nucleic acid in the test sample and the second nucleic acid base in the nucleic acid complex are complementary and pair to form a double-stranded structure.

[0122] PCR reaction is carried out under the action of DNA polymerase, which causes the second nucleic acid to be cleaved and extended using the first nucleic acid as a template, thereby hydrolyzing the third nucleic acid that is complementary to the first nucleic acid;

[0123] (s3) Perform melting curve analysis to obtain nucleic acid detection results;

[0124] The first nucleic acid has the structure shown in formula (I) from 5' to 3':

[0125] B1-B2(I)

[0126] In the formula, B1 consists of 10 to 30 nucleotides;

[0127] B2 consists of 15–30 nucleotides;

[0128] The second nucleic acid has the structure shown in formula (II) from 5' to 3':

[0129] J1-J2(II)

[0130] In the formula, J1 and B2 are complementary;

[0131] J2 is complementary to the target nucleic acid;

[0132] The third nucleic acid is complementary to B1.

[0133] In a preferred embodiment, the third nucleic acid is modified with a fluorescent group and a quenching group.

[0134] In a preferred embodiment, the molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1–10:1–5:1–10, more preferably 1–8:1–3:1–8, and even more preferably 1–5:1–3:1–5, for example, 2:1:2.

[0135] In a preferred embodiment, the premix further includes upstream and downstream primers for amplifying the target nucleic acid.

[0136] In a preferred embodiment, the method includes the steps of: providing a positive standard and performing melting curve analysis on the positive standard to obtain the melting curve height H2 of the positive standard.

[0137] In a preferred embodiment, if the melting curve height H2 of the positive standard decreases by more than 50% compared to the melting curve height H0 of the negative standard, i.e. (H0-H2) / H0>50%, it indicates that the target nucleic acid is present in the positive standard, and the method is effective; if the melting curve height H2 of the positive standard does not decrease by more than 50%, it indicates that the method is ineffective and retesting is required (quality control method).

[0138] The principle of the detection method of this invention is as follows: Figure 1 and 2 As shown. 1) In the detection system, the detection probe, tag sequence, and signal probe will complementarily pair to form a composite structure. The signal probe will change from a single-stranded state to a double-stranded state. Because double-stranded DNA is more rigid than single-stranded DNA, the average distance between the luminescent and quenching groups in the double-stranded state is greater than that in the single-stranded state. Therefore, the fluorescence value in the double-stranded state is higher than that in the single-stranded state. As the temperature rises, the composite structure will change from a double-stranded state to a single-stranded state, and the fluorescence value in the entire system will decrease. The presence of the composite structure is determined by detecting the fluorescence value that changes with temperature in the system. 2) An enzyme is added to the system to remove the J2 region of the detection probe. The 3' end blockage of the J1 region of the detection probe will be eliminated. The J1 region of the detection probe will extend using the tag sequence as a template and hydrolyze the signal probe that is complementary to the B1 region of the tag sequence, thus eliminating the phenomenon of temperature-dependent fluorescence value changes. 3) When a DNA template and DNA polymerase that are complementary to the J2 region of the detection probe are added to the system, based on the mechanism described in 2), the DNA template and the J2 region of the detection probe are complementary, the DNA polymerase cuts the J2 region and hydrolyzes the signal probe, so that the phenomenon of fluorescence value change dependent on temperature change will disappear.

[0139] In summary, in a detection system comprising a detection probe, a tag sequence, and a signal probe (signal probes modified with different fluorescent groups at different melting temperatures), the addition of a DNA template (target nucleic acid) cleaves the J2 region of the detection probe, eliminating the 3' end block of the J1 region. Consequently, the J1 region of the detection probe extends using the tag sequence as a template, hydrolyzing the signal probe and eliminating the temperature-dependent fluorescence change phenomenon. The type of DNA template can be determined by observing the fluorescence signals of different types at different melting temperatures.

[0140] Nucleic acid testing system

[0141] The nucleic acid detection system of the present invention comprises:

[0142] (a) The first nucleic acid, which has the structure shown in formula (I) from 5' to 3':

[0143] B1-B2(I)

[0144] In the formula, B1 consists of 10 to 30 nucleotides;

[0145] B2 consists of 15–30 nucleotides;

[0146] (b) A second nucleic acid, which has the structure shown in formula (II) from 5' to 3':

[0147] J1-J2(II)

[0148] In the formula, J1 and B2 are complementary;

[0149] J2 is complementary to the target nucleic acid; and

[0150] (c) A third nucleic acid, which is complementary to B1.

[0151] In a preferred embodiment, in the nucleic acid detection system, the first nucleic acid, the second nucleic acid, and the third nucleic acid form a nucleic acid complex through complementary base pairing.

[0152] In a preferred embodiment, the nucleic acid detection system further comprises a DNA polymer and / or a PCR reaction solution. In a preferred embodiment, the PCR reaction solution comprises 10× PCR buffer, 0.05–1 mM dNTP, and 1–5 mM MgCl2.

[0153] In a preferred embodiment, the nucleic acid detection system further includes primers for amplifying the target nucleic acid.

[0154] In a preferred embodiment, in the nucleic acid detection system, the molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1–10:1–5:1–10, more preferably 1–8:1–3:1–8, and even more preferably 1–5:1–3:1–5, for example, 2:1:2.

[0155] In a preferred embodiment, the Tm value of the melting curve of the method varies considerably. When designing primers, probes, detection probes, and tag sequences, the Tm value of their melting curves can be selected between 20-70℃, preferably 30-70℃.

[0156] In a preferred embodiment, the primers include an upstream primer and a downstream primer, the concentrations of which in the nucleic acid detection system are independently 50–500 nM, preferably 100–400 nM, more preferably 150–300 nM, for example, about 200 nM. Appropriate primer concentrations can avoid interference between primers and probes and the lower extension temperature required for hybridization of the detection probe, thus avoiding a decrease in sensitivity caused by the amplification product having a Tm value higher than the limited pairing of probes.

[0157] In a preferred embodiment, the concentration of the first nucleic acid in the nucleic acid detection system is 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0158] In a preferred embodiment, the concentration of the second nucleic acid in the nucleic acid detection system is 1 nM to 300 mM, more preferably 10 nM to 50 mM, and even more preferably 20 to 500 nM, for example, about 50 nM.

[0159] In a preferred embodiment, the concentration of the third nucleic acid in the nucleic acid detection system is 1 nM to 500 mM, more preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example about 100 nM.

[0160] In a preferred embodiment, the amount of DNA polymerase is 0.5–5 U, more preferably 0.5–3 U, more preferably 0.5–2 U, for example, about 1 U.

[0161] Nucleic acid test kit

[0162] The nucleic acid detection kit of the present invention comprises:

[0163] (i) A first container and a nucleic acid detection system of the present invention located within the first container.

[0164] In a preferred embodiment, the kit further comprises:

[0165] (ii) The second container and the DNA polymerase located within the second container;

[0166] (iii) the third container and the PCR reaction solution located within the third container; and / or

[0167] (iv) The fourth container and the primers located within the fourth container.

[0168] In a preferred embodiment, the first container, the second container, the third container, and / or the fourth container are the same or different containers.

[0169] The main advantages of this invention include:

[0170] 1. The method of the present invention can detect multiple targets simultaneously in the same reaction tube, with good specificity.

[0171] 2. The method of the present invention is simple in operation, low in cost, fast and accurate.

[0172] 3. The melting peak of the method of the present invention originates from the probe and is not affected by target variation.

[0173] 4. The method of the present invention uses common DNA polymerase, which reduces the difficulty of obtaining raw materials.

[0174] 5. The method of the present invention provides a more digital interpretation of results, making it easier to automate.

[0175] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0176] Example 1

[0177] 1. Primer and probe design

[0178] Studies have shown that HPV genotypes exhibit both homology and specificity, allowing for the design of degenerate primers based on homology sequences. The primer and probe design principle for detecting four HPV types in this invention is as follows: forward and reverse primers are designed using sequences with high homology to each HPV genotype, while probe sequences are designed using sequences with high specificity. PCR is performed using a single fluorescent channel in the same tube, and detection probes are used to detect all four HPV types simultaneously. The upstream primer, downstream primer, and detection probe sequences are shown in Table 1.

[0179] Table 1 Primer and probe sequences for HPV detection

[0180]

[0181]

[0182] Note: In the table, "-" is used to divide the J1 and J2 regions in the detection probe.

[0183] 2. Signal probe and tag sequence design

[0184] To enable the simultaneous detection of four HPV types using a single fluorescence channel in the same reaction tube, the signal probes and tag sequences need to be screened and validated. The signal probes and tag sequences are shown in Tables 2a and 2b.

[0185] Table 2a Sequences of Signal Probes

[0186] SEQ ID NO: sequence name nucleotide sequence 5'-3' 7 Signal probe 1 FAM-TGGTAGGGAACTCGTTT-BHQ1 8 Signal probe 2 FAM-ATCGGTTGTTGTTCTTTT-BHQ1 9 Signal probe 3 FAM-AGCTCCTATTGCCAACGT-BHQ1 10 Signal probe 4 FAM-TACCTTGATGGTCAGCAA-BHQ1

[0187] Table 2a Sequences of Signal Probes

[0188]

[0189] Note: In the table, "-" is used to divide the signal probe into regions B1 and B2.

[0190] 3. System

[0191] This invention employs a non-ionic detergent in conjunction with a high-temperature environment to lyse the HPV outer membrane proteins, releasing the DNA. Simultaneously, Chelex-100 is used to remove impurities such as metal ions that inhibit PCR, thereby improving detection efficiency. To enable the simultaneous detection of four HPV types using a single fluorescence channel in the same reaction tube, the primer and probe concentrations and reaction system must be optimized to avoid interference between primers and probes and to meet the lower extension temperatures required for detection probe hybridization. The optimized PCR system and amplification reaction procedure are shown in Tables 3a and 3b.

[0192] Table 3a PCR system

[0193]

[0194]

[0195] Table 3b PCR reaction procedure

[0196]

[0197] 4. Melting curve analysis

[0198] To enable the simultaneous detection of four HPV types using a single fluorescence channel in the same reaction tube, the melting curves of the signal probes were analyzed using a quantitative real-time PCR instrument. The reaction procedure for melting curve analysis is shown in Table 4.

[0199] Table 4 Reaction Procedure for Melting Curve Analysis

[0200]

[0201] 5. Results

[0202] To enable the simultaneous detection of four HPV types using a single fluorescence channel in the same reaction tube, the primer, probe, detection probe, and tag sequences were designed to achieve melting temperatures of 50.5±1.0℃, 57.0±1.0℃, 33.5±1.0℃, and 42.0±1.0℃ for HPV16, HPV31, HPV39, and HPV66, respectively. This larger temperature interval reduces mutual interference between different types and lowers the probability of false positives.

[0203] like Figure 3 As shown, the method of the present invention can simultaneously detect four types of HPV using a single fluorescence channel.

[0204] Example 2

[0205] 1. Primer and probe design

[0206] Download and compare influenza A, influenza B, respiratory syncytial virus (RSV), and adenovirus from the NCBI database. Design forward and reverse primers based on sequences with high genomic homology, and design probe sequences based on sequences with high specificity. Perform PCR in the same tube using a single fluorescent channel, and simultaneously use detection probes to detect influenza A, influenza B, RSV, and adenovirus. The primer and detection probe sequences used in this example are shown in Table 5.

[0207] Table 5 Primer and detection probe sequences

[0208]

[0209]

[0210] 2. Signal probe and tag sequence design

[0211] In this embodiment, a single fluorescent channel is used to simultaneously detect influenza A virus, influenza B virus, respiratory syncytial virus and adenovirus. The tag sequence and signal probe used in this embodiment are as described in Example 1.

[0212] 3. Reaction system

[0213] This invention employs a magnetic bead method to extract RNA / DNA from influenza A, influenza B, respiratory syncytial virus (RSV), and adenovirus. To enable simultaneous detection of these viruses using a single fluorescence channel in the same reaction tube, the primer / probe concentrations and reaction system must be optimized to avoid interference between primers / probes and to meet the low extension temperatures required for probe hybridization. The optimized PCR system and amplification reaction procedure are shown in Tables 6a and 6b.

[0214] Table 6a PCR system

[0215]

[0216]

[0217] Table 6b PCR reaction procedure

[0218]

[0219] 4. Melting curve analysis

[0220] To enable the simultaneous detection of influenza A, influenza B, respiratory syncytial virus, and adenovirus using a single fluorescence channel in the same reaction tube, the melting curves of the signal probes were analyzed using a quantitative real-time PCR instrument. The reaction procedure for melting curve analysis is shown in Table 4.

[0221] Table 7 Reaction Procedure for Melting Curve Analysis

[0222]

[0223] 5. Result Judgment

[0224] To enable the simultaneous detection of influenza A, influenza B, respiratory syncytial virus, and adenovirus using a single fluorescence channel in the same reaction tube, the melting curve temperatures of the four viruses were found to be 41.8±1.0℃, 35.0±1.0℃, 48.7±1.0℃, and 57.0±1.0℃, respectively. The significant temperature differences resulted in very low false-positive rates due to mutual interference. Furthermore, comparing the melting curve heights in each result with those in the negative control showed a decrease in melting curve height exceeding 20%.

[0225] like Figure 4 As shown, the method of the present invention can simultaneously detect influenza A virus, influenza B virus, respiratory syncytial virus and adenovirus using a single fluorescence channel.

[0226] Example 3

[0227] 1. Primer and probe sequences

[0228] Conventional multiplex detection methods based on melting curves rely on the fact that HPV genotypes exhibit both homology and specificity, allowing for the design of degenerate primers based on homologous sequences. The primer and probe design principle for detecting four HPV types is as follows: forward and reverse primers are designed using sequences with high homology to each HPV genotype, while probe sequences are designed using sequences with high specificity. PCR is performed in the same tube using a single fluorescent channel, and detection probes are used to detect all four HPV types simultaneously. The upstream primer, downstream primer, and detection probe sequences are shown in Table 8.

[0229] Table 8 Primer and probe sequences for HPV detection

[0230] SEQ ID NO: sequence name nucleotide sequence 5'-3' 1 upstream degenerate primers GCMCAGGGWCATAAYAATGG 2 Downstream degenerate primers GAAAAATAWACTGMAAATCATY 27 Probe 1 (HPV16) FAM-TGTGCTGCCTTATTTACTTC-3'BHQ 28 Probe 2 (HPV31) FAM-TATGTCTGTTTGTGCTGC-3'BHQ 29 Probe 3 (HPV39) FAM-GTGTTCTGCTGTGTCTTC-3'BHQ 30 Probe 4 (HPV66) FAM-AAAAACACATTAACTAA-3'BHQ

[0231] 2. System

[0232] This invention employs a non-ionic detergent in conjunction with a high-temperature environment to lyse the HPV outer membrane proteins, releasing the DNA. Simultaneously, Chelex-100 is used to remove impurities such as metal ions that inhibit PCR, thereby improving detection efficiency. To enable the simultaneous detection of four HPV types using a single fluorescence channel in the same reaction tube, the primer and probe concentrations and reaction system must be optimized to avoid sensitivity loss due to the amplification product's Tm value being higher than the probe's preferential pairing. The optimized primer and probe concentrations and reaction system are shown in Table 9a. The amplification reaction procedure in this embodiment is shown in Table 9b.

[0233] Table 9a PCR System

[0234]

[0235] Table 9b PCR reaction procedure

[0236]

[0237] 3. Melting curve analysis

[0238] To enable the simultaneous detection of four HPV types using a single fluorescence channel in the same reaction tube, the melting curves of the signal probes were analyzed using a quantitative real-time PCR instrument. The reaction procedure for melting curve analysis is shown in Table 10.

[0239] Table 10 Reaction Procedure for Melting Curve Analysis

[0240]

[0241] 4. Results

[0242] In order to detect four HPV types simultaneously using a single fluorescence channel in the same reaction tube, the melting curve height in each result was compared with the melting curve height in the negative control. A change in melting curve height of more than 10% was considered positive.

[0243] Example 4 Sample Detection

[0244] Using plasmid samples 16, 31, 39, and 66, detection was performed using the methods described in Example 1 (the method of the present invention) and Example 3 (the method of the prior art).

[0245] The results are shown in Table 11.

[0246] Table 11

[0247]

[0248]

[0249] The results are shown in Table 11. When the target is 100 copies, existing detection methods cannot detect plasmids 31 and 66, resulting in false negatives, while the method of this invention can detect both. The method of this invention can detect HPV as low as 25 copies. High specificity is still maintained even without a target control.

[0250] Table 12

[0251]

[0252]

[0253] As shown in Table 12, the Rm values ​​of Tm corresponding to each target melting peak show a decreasing percentage decrease in melting curve height as the sample concentration decreases. At high concentrations (10,000 copies), the decrease is as high as 95.08%, i.e., (216.03-10.62) / 216.03 = 95.08%; at low concentrations (25 copies), the decrease is as low as 10%, i.e., (146.54-131.82) / 146.54 = 10.04%, which maintains the sensitivity within a good range. Considering equipment and operational errors, a decrease in melting curve height of at least 10% will result in better sensitivity and discrimination.

[0254] To ensure sensitivity for multiple subtypes, the proportion of H1 can be increased, for example, by more than 20%, 30%, 40%, 50%, 60%, or even 70%, 80%, 90%, depending on the characteristics of multiple subtypes in different projects.

[0255] Compared with existing technologies, the multiple detection method of this invention enables closed-tube detection, eliminating the need for steps such as opening the tube for hybridization and color development after PCR, significantly shortening the operation time, reducing the use of supporting equipment and consumables, and reducing the pollution problem caused by high-concentration PCR products.

[0256] The results show that the method described in this invention has higher sensitivity than conventional detection methods, solves the problem of asymmetric PCR affecting detection sensitivity, and has advantages such as low cost, fewer operation steps, and fewer instruments required, demonstrating good development potential. Furthermore, the method of this invention can be used on most real-time PCR instruments, achieving multiplex detection capabilities without the need for additional instruments, resulting in better economic benefits.

[0257] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nucleic acid detection method, characterized in that, The method includes the following steps: (s1) Provides a premix, a sample to be tested, and a DNA polymerase, wherein the premix contains a nucleic acid complex formed by a first nucleic acid, a second nucleic acid, and a third nucleic acid through complementary base pairing; (s2) The test sample is mixed with the premix and DNA polymerase, so that the target nucleic acid in the test sample and the second nucleic acid base in the nucleic acid complex are complementary and pair to form a double-stranded structure. PCR reaction is carried out under the action of DNA polymerase, which causes the second nucleic acid to be cleaved and extended using the first nucleic acid as a template, thereby hydrolyzing the third nucleic acid that is complementary to the first nucleic acid; (s3) Perform melting curve analysis to obtain nucleic acid detection results; The first nucleic acid has the structure shown in formula (I) from 5' to 3': B1-B2(I) In the formula, B1 consists of 10 to 30 nucleotides; B2 consists of 15–30 nucleotides; The second nucleic acid has the structure shown in formula (II) from 5' to 3': J1-J2(II) In the formula, J1 and B2 are complementary; J2 is complementary to the target nucleic acid; The third nucleic acid is complementary to B1.

2. The method as described in claim 1, characterized in that, The third nucleic acid modification includes a fluorescent group and a quenching group.

3. The method as described in claim 1, characterized in that, B1 consists of 15–30 nucleotides, preferably 15–25 nucleotides, and even more preferably 15–23 nucleotides.

4. The method as described in claim 1, characterized in that, B2 consists of 15–25 nucleotides, preferably 15–23 nucleotides, and even more preferably 16–23 nucleotides.

5. The method as described in claim 1, characterized in that, In step (s1), the molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1-10:1-5:1-10, preferably 1-8:1-3:1-8, more preferably 1-5:1-3:1-5, for example, 2:1:

2.

6. The method as described in claim 1, characterized in that, The premix also includes upstream and downstream primers for amplifying the target nucleic acid.

7. A nucleic acid detection system, characterized in that, The nucleic acid detection system includes: (a) The first nucleic acid, which has the structure shown in formula (I) from 5' to 3': B1-B2(I) In the formula, B1 consists of 10 to 30 nucleotides; B2 consists of 15–30 nucleotides; (b) A second nucleic acid, which has the structure shown in formula (II) from 5' to 3': J1-J2(II) In the formula, J1 and B2 are complementary; J2 is complementary to the target nucleic acid; and (c) A third nucleic acid, which is complementary to B1.

8. A nucleic acid detection kit, characterized in that, The kit contains: (i) The first container and the nucleic acid detection system as described in claim 7 located within the first container.

9. A nucleic acid detection method, characterized in that, Including the following steps: After performing nucleic acid amplification reaction on the sample to be tested using the nucleic acid detection system as described in claim 7 or the detection kit as described in claim 8, melting curve analysis is performed to obtain the detection results.

10. The use of a nucleic acid detection system as described in claim 7 or a detection kit as described in claim 8, characterized in that, Used to detect nucleic acids in samples to be tested.

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