Method and kit for detecting target nucleic acid by melting curve

Abstract

The invention provides a method for detecting target nucleic acid through a melting curve and a kit thereof. The method comprises the following steps: designing a specific upstream primer, a downstream primer and a tag probe aiming at a target nucleic acid sequence; designing a detection probe aiming at the tag sequence, modifying a quenching group on the detection probe, performing amplification in an amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe and nucleic acid to be detected, and performing melting curve detection on a double-chain product to obtain whether the detected nucleic acid to be detected contains target nucleic acid or not. According to the present invention, with the newly designed label probe and the newly designed detection probe, the interference fluorescence signal fluctuation in the melting curve analysis process is reduced, the fluorescence signal fluctuation of the label and the detection probe is represented in the melting curve analysis process in the large proportion, the straight baseline is obtained, the non-specific peak occurrence is reduced, and the detection accuracy is improved; and the accuracy and accuracy of the detection method are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly to a method for detecting target nucleic acid by melting curve and a kit thereof. Background Art

[0002] Real-time fluorescence PCR technology refers to adding a fluorescent group to the PCR reaction system. During the PCR amplification process, the PCR progress is detected in real time through the fluorescent signal. It is the most widely used detection method for nucleic acid detection. It has the characteristics of simple operation, fast and efficient, high throughput, and high sensitivity. This technology has a wide range of applications in molecular diagnosis, molecular biology research, animal and plant quarantine, and food safety detection.

[0003] Real-time fluorescence PCR can use a fluorescent probe as the fluorescent substance. The fluorescent probe is modified with a fluorescent group and a quenching group at both ends of the oligonucleotide. The fluorescent probe can specifically bind to the target. Multiplex nucleic acid detection often uses multiplex real-time fluorescence PCR, and oligonucleotides are modified with different fluorescent groups. In the real-time fluorescence PCR method, the target sequence can be detected through two modes, namely the real-time detection mode and the melting curve analysis after amplification. In the real-time detection mode, the detection of the target sequence and the PCR amplification are carried out simultaneously without the need for additional steps. Therefore, the real-time detection mode is simple and direct. However, the maximum number of target sequences that can be detected in a single-round detection in this mode is limited by the number of fluorescent detection channels of the real-time fluorescence quantitative PCR instrument, generally not exceeding 6. In the melting curve mode, an additional step is required after PCR amplification, that is, the melting curve analysis of the probe and the target sequence amplification product. The melting curve can identify or distinguish the target sequence through the fluorescent color and / or melting point. Therefore, although the melting curve mode is relatively time-consuming, that is, an additional step is added, the maximum number of target sequences that can be detected in a single-round detection is greatly improved.

[0004] Fluorescent probe multiplex PCR technology is based on real-time fluorescence PCR technology. By using a combination of several different fluorescent groups and combining the instrument's ability to detect fluorescence in different channels, the detection of multiple targets is achieved. For the multiplex nucleic acid detection of fluorescent probe multiplex PCR technology, in order to distinguish the results of different targets, the probes corresponding to different targets need to be labeled with different fluorescences. Limited by the number of known fluorescent groups and the number of channels readable by the instrument, there is a limit to the detection multiplicity. The current technology generally does not exceed 6-plex. When detecting multiple targets, it is necessary to detect in multiple wells, which increases the labor and material costs and reduces the detection throughput.

[0005] CN108823287A discloses a multiplex real-time fluorescence PCR method that can be used for melting curve analysis. In this method, the detection of the target sequence is achieved through mediator probes and detection probes. The mediator probe contains a mediator sequence and a target-specific sequence. The detection probe contains multiple capture sequences that are complementary to multiple mediator sequences or parts thereof. The detection probe is a self-quenching probe and contains a reporter group and a quenching group. During the detection process, the mediator probe binds to the target nucleic acid, the mediator probe is cleaved to release the mediator sequence, the mediator sequence is captured by the detection probe and extended, and the extended product is subjected to melting curve analysis. In the method disclosed in CN108823287A, since the detection probe contains a reporter group and a quenching group, when the melting curve analysis is performed on the extended product of the mediator sequence and the detection probe, the phenomenon of uneven baseline and non-specific peak detection is likely to occur, resulting in misjudgment of the detection result. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method for detecting a target nucleic acid by melting curve, a method for detecting a target nucleic acid by melting curve of a double-stranded product formed by complementary extension of a tag sequence extension product and a detection probe, and the method includes the following steps:

[0007] Step 1: Design specific upstream primers, downstream primers, and tag probes for the target nucleic acid sequence. The upstream primer and the downstream primer specifically bind to the target nucleic acid. The tag probe includes a 5'-end tag sequence and a 3'-end target-specific sequence. The tag sequence cannot bind to the target nucleic acid, the tag sequence is modified with a fluorescent group, the target-specific sequence can specifically bind to the target nucleic acid, the target-specific sequence is modified with a quenching group, and the quenching group on the 3'-end of the tag probe quenches the fluorescent group on the tag sequence.

[0008] Step 2: Design a detection probe for the 5'-end tag sequence. The detection probe is modified with a quenching group, and the number of the modified quenching groups is ≥1. The detection probe has a sequence that can be complementary to the tag sequence and can capture the tag sequence.

[0009] Step 3: Amplification is carried out in a PCR amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe, the nucleic acid to be tested, and Taq enzyme. If the nucleic acid to be tested contains the target nucleic acid, both the upstream primer and the downstream primer are extended. When the extension reaches the position of the tag probe, Taq enzyme cuts off the tag sequence. The cut-off tag sequence is captured by the detection probe and extended. The extended product of the tag sequence and the detection probe complementarily form a double-stranded product. At this time, the quenching group on the detection probe quenches the fluorescent group on the tag sequence. As the amplification reaction proceeds, the gradually increasing number of cut-off tag probes by Taq enzyme, and the increasing number of tag sequences captured by the detection probe and extended, finally a double-stranded product for melting curve detection is obtained.

[0010] Step 4: Melting curve detection is performed on the double-stranded product obtained in Step 3 to determine whether the nucleic acid to be tested contains the target nucleic acid.

[0011] In one embodiment, 4 - 30 bp at the 5'-end of the tag probe is the tag sequence.

[0012] In one embodiment, 10 - 40 bp at the 3'-end of the tag probe is the target-specific sequence.

[0013] In one embodiment, the modification at the 3'-end of the tag probe to prevent extension is phosphate modification.

[0014] In one embodiment, a method for detecting a target nucleic acid by melting curve is provided. The method for detecting a target nucleic acid by melting curve through the double-stranded product formed by the complementarity of the extended product of the tag sequence and the detection probe includes the following steps:

[0015] Step 1: Design specific upstream primer, downstream primer, and tag probe for the target nucleic acid sequence. The upstream primer and the downstream primer specifically bind to the target nucleic acid. The tag probe includes a 5'-end tag sequence and a 3'-end target-specific sequence. The 5'-end tag sequence includes the front part and the back part of the 5'-end tag. The front part can bind to the detection probe and cannot bind to the target nucleic acid. The back part cannot bind to the detection probe and the target nucleic acid and plays a role in preventing extension. The tag sequence is modified with a fluorescent group. The target-specific sequence can specifically bind to the target nucleic acid. There is a quenching group on the target-specific sequence. The 3'-end of the tag probe is modified with a group that prevents extension. The quenching group on the target-specific sequence quenches the fluorescent group on the tag sequence.

[0016] Step 2: Design a detection probe for the 5'-end tag sequence. The detection probe is modified with a quenching group, and the number of modified quenching groups is ≥1. There is a sequence on the detection probe that can be complementary to the tag sequence and can capture the tag sequence.

[0017] Step 3: Perform amplification in a PCR amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe, the nucleic acid to be tested, and Taq enzyme. If the nucleic acid to be tested contains the target nucleic acid, both the upstream primer and the downstream primer are extended. When the extension reaches the position of the tag probe, Taq enzyme cuts off the tag sequence. The cut-off tag sequence is captured by the detection probe, and the cut-off tag sequence and the detection probe are complementary to form a double-stranded product. At this time, the quenching group on the detection probe quenches the fluorescent group on the tag sequence. As the amplification reaction proceeds, the number of tag probes cut off by Taq enzyme gradually increases, and the number of tag sequences captured and cut off by the detection probe increases. Finally, a double-stranded product for melting curve detection is obtained.

[0018] Step 4: Perform melting curve detection on the double-stranded product obtained in Step 3 to determine whether the nucleic acid to be tested contains the target nucleic acid.

[0019] In one embodiment, the length of the front part of the 5'-end tag of the 5'-end tag sequence is 4 - 30 bp, and the length of the rear part of the 5'-end tag of the 5'-end tag sequence is 3 - 5 bp.

[0020] In one embodiment, the length of the 3'-end target-specific sequence is 10 - 40 bp.

[0021] In one embodiment, the 3'-end modification of the tag probe to prevent extension is a phosphate modification.

[0022] In one embodiment, a method for detecting a target nucleic acid by melting curve is provided. The method for detecting a target nucleic acid by melting curve through a double-stranded product formed by the complementarity of a tag sequence extension product and a detection probe includes the following steps:

[0023] Step 1: Design a specific upstream primer, a downstream primer, and a tag probe for the target nucleic acid sequence. The upstream primer and the downstream primer specifically bind to the target nucleic acid. The tag probe includes a 5'-end tag sequence and a 3'-end target-specific sequence. The tag sequence cannot bind to the target nucleic acid, and the tag sequence is modified with a fluorescent group. The target-specific sequence can specifically bind to the target nucleic acid. The first to fifth bases of the target-specific sequence are one or more consecutive RNA bases, and the target-specific sequence is modified with a quenching group. The quenching group on the target-specific sequence quenches the fluorescent group on the tag sequence. The 3'-end of the tag probe is modified with a group that prevents extension.

[0024] Step 2: Design a detection probe for the 5'-end tag sequence. Modify a quenching group on the detection probe, and the number of modified quenching groups is ≥1. There is a sequence on the detection probe that can be complementary to the tag sequence, and the tag sequence can be captured.

[0025] Step 3: Perform amplification in a PCR amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe, the nucleic acid to be tested, RNase H enzyme, and Taq enzyme. If the nucleic acid to be tested contains the target nucleic acid, the upstream primer, the downstream primer, and the tag probe all bind to the target to be tested during annealing. The upstream primer and the downstream primer both extend. The target-specific sequence of the tag probe binds to the target nucleic acid to form a DNA / RNA hybrid strand. The RNase enzyme can specifically hydrolyze the phosphodiester bond on the DNA / RNA hybrid strand to obtain the hydrolyzed tag sequence. The hydrolyzed tag sequence is captured by the detection probe and extended. The extended product of the tag sequence and the detection probe are complementary to form a double-stranded product. At this time, the quenching group on the detection probe quenches the fluorescent group on the tag sequence. As the amplification reaction proceeds, the hydrolyzed tag probes by the RNase enzyme gradually increase, and the tag sequences captured by the detection probe and extended increase. Finally, a double-stranded product for melting curve detection is obtained.

[0026] Step 4: Perform melting curve detection on the double-stranded product obtained in Step 3 to obtain whether the nucleic acid to be tested contains the target nucleic acid.

[0027] In one embodiment, 4 - 30 bp at the 5'-end of the tag probe is the tag sequence.

[0028] In one embodiment, a method for detecting a target nucleic acid by melting curve is provided. 10 - 40 bp at the 3'-end of the tag probe is the target-specific sequence.

[0029] In one embodiment, a method for detecting a target nucleic acid by melting curve is provided. The modification at the 3'-end of the tag probe to prevent extension is a phosphate modification.

[0030] In one embodiment, the present invention provides a PCR kit for detecting a target nucleic acid based on melting curve, and the kit is applied in the above method.

[0031] In one embodiment, the present invention provides a multiplex PCR kit for detecting multiple target nucleic acids based on melting curves. The multiplex PCR kit detects target nucleic acids through the melting curves of double-stranded products formed by the complementarity between the tag sequence extension products and the detection probes. The kit includes specific upstream primers, downstream primers, and tag probes designed for the target nucleic acid sequences. The upstream primers and downstream primers specifically bind to the target nucleic acid. The tag probes are selected from one or more of the following three tag probes:

[0032] The first tag probe, which includes a 5'-end tag sequence and a 3'-end target-specific sequence. The tag sequence cannot bind to the target nucleic acid, and the tag sequence is modified with a fluorescent group. The target-specific sequence can specifically bind to the target nucleic acid, and the target-specific sequence is modified with a quenching group. The 3'-end of the tag probe is modified with a group that blocks extension. The quenching group on the target-specific sequence quenches the fluorescent group on the tag sequence.

[0033] The second tag probe, which includes a 5'-end tag sequence and a 3'-end target-specific sequence. The 5'-end tag sequence includes a front part and a rear part of the 5'-end tag. The front part can bind to the detection probe and cannot bind to the target nucleic acid. The rear part cannot bind to the detection probe and the target nucleic acid and plays a role in blocking extension. The tag sequence is modified with a fluorescent group. The target-specific sequence can specifically bind to the target nucleic acid, and the target-specific sequence is modified with a quenching group. The 3'-end of the tag probe is modified with a group that blocks extension. The quenching group on the target-specific sequence quenches the fluorescent group on the tag sequence.

[0034] The third tag probe, which includes a 5'-end tag sequence and a 3'-end target-specific sequence. The tag sequence cannot bind to the target nucleic acid, and the tag sequence is modified with a fluorescent group. The target-specific sequence can specifically bind to the target nucleic acid. The 1st to 5th bases of the target-specific sequence are one or more consecutive RNA bases. The target-specific sequence is modified with a quenching group. The 3'-end of the tag probe is modified with a group that blocks extension. The quenching group on the target-specific sequence quenches the fluorescent group on the tag sequence.

[0035] The detection probe is designed for the 5'-end tag sequence. The detection probe is modified with a quenching group, and the number of modified quenching groups is ≥1. There is a sequence on the detection probe that can be complementary to the tag sequence and can capture the tag sequence.

[0036] When the third tag probe is not selected, the kit further includes Taq enzyme. When the third tag probe is selected, the kit includes both Taq enzyme and RNase enzyme.

[0037] In one embodiment, the labeled probe is selected from a first labeled probe and a second labeled probe.

[0038] The present invention provides a method for detecting a target nucleic acid by melting curve. By newly designed labeled probes and detection probes, the situation of interfering fluorescence signal fluctuations during the melting curve analysis is reduced. A larger proportion of the melting curve analysis process reflects the fluorescence signal fluctuations of the labeled and detection probes, obtaining a flatter baseline, reducing the appearance of non-specific peaks, and improving the accuracy and correctness of the detection method. At the same time, this method is not limited by the number of fluorescence channels detected. Different targets are distinguished according to the melting point and fluorescence color, increasing the number of targets detected in a single well and improving the detection throughput. With the method of the present invention, multiple target detections can be performed. The baselines of the melting peaks can all remain flat, the peaks are higher, and there are no non-specific peaks. Nucleic acids to be detected with a concentration of 10 copies / μL can all be detected simultaneously, that is, it has higher sensitivity. At the same time, the detection interval range is also wider. The Tm value range for melting curve detection is: 20-90 °C. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the melting curve graph of the first labeled probe of the present invention for detecting HPV16;

[0040] Figure 2 It is the melting curve graph of detecting HPV16 by the conventional probe method;

[0041] Figure 3 It is the melting curve graph of the second labeled probe of the present invention for detecting HPV51;

[0042] Figure 4 It is the melting curve graph of the third labeled probe of the present invention for detecting HPV56;

[0043] Figure 5 It is the melting curve graph of detecting HPV16, HPV26, HPV51 and HPV45 by the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] In order to enable those skilled in the art to better understand the technical solutions in the present application, the present invention will be further described below in conjunction with embodiments. Obviously, the embodiments are only examples and cannot limit the present invention. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present application. The experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained from commercial channels.

[0045] Example 1 Single-target Detection Experiment of the First Labeled Probe of the Present Invention

[0046] Taking the detection of HPV16 (human papillomavirus type 16) as an example, the method of the present invention and the melting curve of the conventional probe method are used for qualitative detection of the HPV16 quality control product. The specific method includes the following steps:

[0047] I. Primer and probe sequence information of HPV16

[0048] The primer and probe sequence information of the method of the present invention: According to the conserved region of the nucleic acid sequence to be detected, upstream primers, downstream primers, tag probes and detection probes are designed. The sequence information is shown in the following table.

[0049]

[0050] Note: The lowercase letter bases are the tag sequences. The capital letters with underlines in the lowercase letters are fluorescent group modifications. The Q with underlines in the capital letters is the quenching group modification. The italicized and underlined ones are the sequences complementary to the tag.

[0051] The primer and probe sequence information of the melting curve of the conventional probe method: According to the conventional melting curve design method of the probe method, upstream primers, downstream primers and detection probes are designed in the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0052]

[0053]

[0054] Note: The lowercase letters indicate the stem regions of the probes.

[0055] II. Amplification system

[0056] The PCR amplification system of the method of the present invention: 0.2 μL of 5U HS Taq DNA Polymerase is added, 2 μL of 10×Buffer is added, 1 μL of 0.25 mM dNTP is added, 0.4 μL of 10 μM upstream and downstream primers are respectively added, 0.2 μL of 10 μM tag probe is added, 0.4 μL of 10 μM detection probe is added, and 5 μL of the nucleic acid to be detected is added. The total volume of the reaction system is 20 μL.

[0057]

[0058] The PCR amplification system of the melting curve of the conventional probe method: 0.2 μL of 5U HS Taq DNA Polymerase is added, 10 μL of 2×Buffer (dNTP) is added, 0.4 μL of 10 μM F2 is added, 1.6 μL of 10 μM R2 is added, 0.2 μL of 10 μM detection probe is added, and 5 μL of the nucleic acid to be detected is added. The total volume of the reaction system is 20 μL.

[0059]

[0060] III. Amplification Program

[0061] The fluorescence PCR reaction program of the method of the present invention: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing and extension at 60°C for 1 min while collecting fluorescence, repeating 50 cycles; the melting curve program is 35°C for 20 min, 95°C for 2 min, 30°C for 5 min, and 30 - 85°C is for melting curve analysis, detecting the fluorescence signal every 0.04°C.

[0062]

[0063]

[0064] The fluorescence PCR reaction program of the conventional probe method for melting curve: The fluorescence PCR reaction program of the method of the present invention: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 58°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C is for melting curve analysis, detecting the fluorescence signal every 0.04°C.

[0065]

[0066] IV. Detection Results

[0067] The detection result of HPV16 by the method of the present invention is as Figure 1 shown. The Tm value of the melting peak is at 70.5°C. The dotted line is the set baseline. The baseline of this result is straight, the peak value meets the expectation, and there is no non-specific peak. The result of detecting HPV16 by the melting curve of the conventional probe method is as Figure 2 shown. The Tm value of the melting peak is at 67°C. The dotted line is the set baseline. The baseline of this result is uneven, resulting in a low peak value and affecting the result interpretation.

[0068] By comparing the method of the present invention with the melting curve of the conventional probe method, better results can be obtained. The baseline of the melting peak is straighter, the peak value is higher, and there is no non-specific peak. Therefore, the detection sensitivity can be significantly improved.

[0069] Example 2 Multiplex Target Detection of the First Type of Label Probe

[0070] Taking the detection of HPV (human papillomavirus) as an example, the method of the present invention is used to qualitatively detect the quality control products of HPV types 16, 18, 52, and 53. The specific method includes the following steps.

[0071] I. Primer and Probe Sequence Information

[0072] Design upstream primers, downstream primers, tag probes, and detection probes according to the conserved regions of the nucleic acid sequences to be tested. The sequence information is shown in the following table.

[0073]

[0074]

[0075] Note: The bases in lowercase letters are the tag sequences. The capital letters in lowercase letters with an underline are fluorescent group modifications. The letter Q in capital letters with an underline is a quenching group modification. The italicized and underlined sequences are complementary to the tags.

[0076] II. Amplification System

[0077] Add 0.2 μL of 5U HS Taq DNA Polymerase, 2 μL of 10× Buffer, 1 μL of 0.25 mM dNTP, 0.4 μL of 10 μM upstream and downstream primers respectively, 0.2 μL of 10 μM tag probes respectively, 0.4 μL of 10 μM detection probes respectively, and add 5 μL of the nucleic acid to be tested. Detect nucleic acids at two concentrations: 100 copies / μL and 10 copies / μL. The total volume of the reaction system is 20 μL.

[0078]

[0079] III. Amplification Program

[0080] Pre-denature at 95°C for 5 min; denature at 95°C for 20 s, anneal and extend at 60°C for 1 min while collecting fluorescence, repeat 50 cycles; the melting curve program is 35°C for 20 min, 95°C for 2 min, 30°C for 5 min, and 30 - 85°C is for melting curve analysis, detecting the fluorescence signal every 0.04°C.

[0081]

[0082] IV. Detection Results

[0083] The Tm values of the melting peaks of HPV types 16, 26, 51, and 45 are: 70.4°C, 70.4°C, 59.7°C, and 61.0°C respectively. The baseline of this result is flat, 4 targets can be detected simultaneously, and the nucleic acids to be tested at 100 copies / μL and 10 copies / μL can both be detected simultaneously. The peak values meet the expectations and there are no non-specific peaks. With the method of the present invention, multiplex target detection can be carried out, the baselines of the melting peaks can all remain flat, the peak values are higher, and there are no non-specific peaks. The nucleic acids to be tested at 10 copies / μL can all be detected simultaneously, that is, it has higher sensitivity.

[0084] Single-target detection of the second tag probe in Example III

[0085] Taking the detection of HPV (human papillomavirus) as an example, the qualitative detection of the type 51 quality control product of HPV is carried out by the method of the present invention, and the specific method includes the following steps.

[0086] I. Primer and probe sequence information

[0087] In this embodiment, the design of the upstream primer, downstream primer and detection probe is the same as that in Example 1, and there are differences in the design of the tag probe.

[0088] The design of the tag probe is divided into two parts, namely the tag sequence and the target-specific sequence. The design of the tag sequence is as follows: there is a fluorescent group modification on the tag sequence. The first 4-30 bp at the 5' end is the front part of the tag, which can bind to the detection probe and cannot bind to the target nucleic acid. The latter part has 3-5 bp that cannot bind to the detection probe and the target nucleic acid and plays a role in blocking extension; the 3-5 bp at the latter part of the tag is an irrelevant sequence and plays a role in blocking extension. The purpose is that when the tag is cut off and binds to the detection probe, no extension occurs. Target-specific sequence: the 3'-terminal 10-40 bp of the tag probe is the target-specific sequence, which can specifically bind to the target nucleic acid. There is a quenching group on the target-specific sequence, and there is a modification for blocking extension at the 3' end, such as phosphate modification, amino modification, etc. When using the second tag probe, a lower Tm value detection can be obtained. Combining Method 1 and Method 2, the detection temperature range is 20-90 °C. If only Method 1 is used, it can only be detected at 40-80 °C.

[0089] In this embodiment, the upstream primer, downstream primer, tag probe and detection probe of the HPV 51 target are designed according to the conserved region of the nucleic acid sequence to be detected, and the sequence information is shown in the following table.

[0090]

[0091] Note: The lowercase letter bases are the tag sequences. The capital letters with underlines in the lowercase letters are the fluorescent group modifications. The italicized and underlined lowercase letters are the settings for blocking the tag sequence verification. The capital letters with "Q" underlined are the quenching group modifications. The italicized and underlined are the sequences complementary to the tag.

[0092] II. Amplification system

[0093] Add 0.2 μL of 5U HS Taq DNA Polymerase, 2 μL of 10× Buffer, 1 μL of 0.25 mM dNTP, 0.4 μL of 10 μM upstream and downstream primers respectively, 0.2 μL of 10 μM tag probe, 0.4 μL of 10 μM detection probe, and add 5 μL of the nucleic acid to be detected. The total volume of the reaction system is 20 μL.

[0094]

[0095] III. Amplification Program

[0096] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing and extension at 60°C for 1 min while collecting fluorescence, repeat 50 cycles; melting curve program is 35°C for 20 min, 95°C for 2 min, 30°C for 5 min, melting curve analysis from 20 - 80°C, detecting fluorescence signal every 0.04°C.

[0097]

[0098]

[0099] IV. Detection Results

[0100] The detection result of HPV51 is as Figure 3 shown. The Tm value of the melting peak is at 29.33°C. The dotted line is the set baseline. The baseline of this result is flat, the peak value meets the expectation, and there is no non-specific peak. Using the method of the present invention, the baseline of the melting peak is flatter, the peak value meets the expectation, and there is no non-specific peak. Therefore, the detection sensitivity can also be improved.

[0101] Example 4 The Third Type of Label Probe Single-Target Detection Experiment of the Present Invention

[0102] Taking the detection of HPV (human papillomavirus) as an example, the method of the present invention is used to qualitatively detect the 56th type of HPV quality control product. The specific method includes the following steps.

[0103] I. Primer and Probe Sequence Information

[0104] The design of the upstream primer, downstream primer and detection probe in this example is the same as that in Example 1, but there are differences in the design of the label probe.

[0105] The design of the label probe is as follows: 4 - 30 bp at the 5' end of the label probe is the label sequence, which cannot bind to the target nucleic acid and is modified with a fluorescent group; 10 - 40 bp at the 3' end of the label probe is the target-specific sequence, which can specifically bind to the target nucleic acid. The first base of the target-specific sequence is RNA-modified, and there is a quenching group on the target-specific sequence. There is a modification to prevent extension at the 3' end, such as phosphate modification, amino modification, etc.

[0106] In this example, the upstream primer, downstream primer, label probe and detection probe are designed according to the conserved region of the HPV56 nucleic acid sequence. The sequence information is shown in the following table.

[0107]

[0108] Note: The lowercase letter bases are the tag sequences, ROX is fluorescent group modification, the dotted underline is RNA modification, the letter with underline in uppercase letters is quencher group modification, and the italicized and underlined is the sequence complementary to the tag.

[0109] II. Amplification system

[0110] Add 0.2 μL of 5 U HS Taq DNA Polymerase, 0.4 μL of RNase H enzyme, 2 μL of 10× Buffer, 1 μL of 0.25 mM dNTP, 0.4 μL of 10 μM upstream and downstream primers respectively, 0.2 μL of 10 μM tag probe, 0.4 μL of 10 μM detection probe, add 5 μL of the nucleic acid to be tested, and the total volume of the reaction system is 20 μL.

[0111]

[0112]

[0113] III. Amplification program

[0114] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20 s, annealing and extension at 60°C for 1 min, while collecting fluorescence, repeat 50 cycles; the melting curve program is 35°C for 20 min, 95°C for 2 min, 30°C for 5 min, and the melting curve analysis is carried out at 30 - 80°C, and the fluorescence signal is detected every 0.04°C.

[0115]

[0116] IV. Detection results

[0117] The detection result of HPV56 is as Figure 4 shown. The Tm value of the melting peak is 70.4°C. The dotted line is the set baseline. The baseline of this result is flat, the peak value meets the expectation, and there is no non-specific peak. Using the method of the present invention, the baseline of the melting peak is flatter, the peak value meets the expectation, and there is no non-specific peak. Therefore, the detection sensitivity can also be improved.

[0118] Example 4 Combined multiple target detection experiment of the present invention

[0119] Taking the detection of HPV (human papillomavirus) as an example, the combination design of the method of the present invention is used to qualitatively detect the quality control products of HPV types 16, 26, 51, and 45. The specific method includes the following steps.

[0120] I. Primer and probe sequence information

[0121] Design using the combination of the method of the present invention. According to the conserved region of the nucleic acid sequence to be detected, upstream primers, downstream primers, tag probes and detection probes are designed, and the sequence information is shown in the following table; in this example, the first tag probe and the second tag probe are selected and used in combination, which can increase the detection temperature range after combination.

[0122]

[0123]

[0124] Note: The lowercase letter bases are tag sequences. The capital letters in lowercase letters with underlines are fluorescent group modifications. The italic lowercase letters with underlines are for preventing tag sequence verification settings. The letters in capital letters with underlines are quencher group modifications. The italic underlined sequences are complementary to the tags.

[0125] III. Amplification system

[0126] Add 0.2 μL of 5U HS Taq DNA Polymerase, 2 μL of 10× Buffer, 1 μL of 0.25 mM dNTP, 0.4 μL of 10 μM upstream and downstream primers respectively, 0.2 μL of 10 μM tag probes respectively, 0.4 μL of 10 μM detection probes respectively, and add 5 μL of the nucleic acid to be detected. Detect nucleic acids at two concentrations: 100 copies / μL and 10 copies / μL. The total volume of the reaction system is 20 μL.

[0127]

[0128]

[0129] IV. Amplification program

[0130] Pre-denature at 95°C for 5 min; denature at 95°C for 20 s, anneal and extend at 60°C for 1 min, and collect fluorescence simultaneously. Repeat 50 cycles; the melting curve program is 35°C for 20 min, 95°C for 2 min, 30°C for 5 min. The melting curve analysis is from 20 - 90°C, and the fluorescence signal is detected every 0.04°C.

[0131]

[0132] V. Detection results

[0133] The detection results of HPV types 16, 26, 51, and 45 are as Figure 5As shown, the melting peak Tm values of HPV types 16, 26, 51, and 45 are 70.4°C, 70.4°C, 29.2°C, and 29.1°C respectively. The dotted line is the set baseline. The baseline of this result is straight, and 4 targets can be detected simultaneously. Moreover, the nucleic acids to be detected with 100 copies / μL and 10 copies / μL can both be detected simultaneously. The peak values meet the expectations and there are no non-specific peaks. At the same time, the detection interval range is also wider, and the Tm value range for melting curve detection is: 20 - 90°C.

[0134] The method of the present invention can be used for multiplex target detection. The baselines of the melting peaks can all remain straight, the peak values are higher, and there are no non-specific peaks. The nucleic acids to be detected with 10 copies / μL can all be detected simultaneously, that is, it has higher sensitivity. There is also a wider detection interval, and the Tm value range for melting curve detection is: 20 - 90°C.

[0135] It should be understood that the present invention disclosed is not limited to the specific methods, schemes, and substances described, as these can vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments of the scheme and are not intended to limit the scope of the present invention. The scope of the present invention is only limited by the appended claims.

[0136] Those skilled in the art will also recognize, or be able to confirm using no more than routine experiments, many equivalents of the specific embodiments of the present invention described herein. These equivalents are also included in the appended claims.

Claims

1. A method for detecting a target nucleic acid by melting curve, characterized in that, A method for detecting a target nucleic acid by melting curve of a double-stranded product formed by complementarity between a tag sequence extension product and a detection probe, the method comprising the following steps: Step 1: Design specific upstream primers, downstream primers and tag probes for the target nucleic acid sequence, the upstream primers and downstream primers specifically binding to the target nucleic acid; the tag probe includes a 5'-end tag sequence and a 3'-end target-specific sequence, the tag sequence cannot bind to the target nucleic acid, the tag sequence is modified with a fluorophore, the target-specific sequence can specifically bind to the target nucleic acid, the target-specific sequence is modified with a quencher group, and the quencher group on the 3'-end of the tag probe quenches the fluorophore on the tag sequence; Step 2: Design a detection probe for the 5'-end tag sequence, the detection probe is modified with a quencher group, and the number of modified quencher groups is ≥1, and there is a sequence on the detection probe that can be complementary to the tag sequence and can capture the tag sequence; Step 3: Perform amplification in a PCR amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe, the nucleic acid to be tested and Taq enzyme. If the nucleic acid to be tested contains the target nucleic acid, both the upstream primer and the downstream primer are extended. When the extension reaches the position of the tag probe, Taq enzyme cuts off the tag sequence, and the cut tag sequence is captured by the detection probe and extended. The tag sequence extension product and the detection probe are complementary to form a double-stranded product. At this time, the quencher group on the detection probe quenches the fluorophore on the tag sequence; as the amplification reaction proceeds, the number of tag probes cut off by Taq enzyme gradually increases, and the number of tag sequences captured by the detection probe and extended increases, and finally a double-stranded product for melting curve detection is obtained; Step 4: Perform melting curve detection on the double-stranded product obtained in Step 3 to obtain whether the nucleic acid to be tested contains the target nucleic acid.

2. The method according to claim 1, characterized in that, The 5'-end 4-30 bp of the tag probe is the tag sequence.

3. The method according to claim 1, wherein The 3'-end 10-40 bp of the tag probe is the target-specific sequence.

4. The method according to claim 1, wherein The modification of the 3'-end of the tag probe to prevent extension is phosphate modification.

5. Method for detecting target nucleic acid by melting curve, characterized in that, A method for detecting a target nucleic acid by melting curve of a double-stranded product formed by complementarity between a tag sequence and a detection probe, the method comprising the following steps: Step 1: Design specific upstream primer, downstream primer and tag probe for the target nucleic acid sequence. The upstream primer and downstream primer specifically bind to the target nucleic acid. The tag probe includes a 5'-end tag sequence and a 3'-end target-specific sequence. The 5'-end tag sequence includes the front part and the rear part of the 5'-end tag. The front part can bind to the detection probe and cannot bind to the target nucleic acid. The rear part cannot bind to the detection probe and the target nucleic acid and plays a role in preventing extension. The tag sequence is modified with a fluorophore. The target-specific sequence can specifically bind to the target nucleic acid. The target-specific sequence is modified with a quencher group. The 3'-end of the tag probe is modified with a group that prevents extension. The quencher group on the target-specific sequence quenches the fluorophore on the tag sequence. Step 2: Design a detection probe for the 5'-end tag sequence. The detection probe is modified with a quencher group, and the number of modified quencher groups is ≥1. There is a sequence on the detection probe that can be complementary to the tag sequence and can capture the tag sequence. Step 3: Perform amplification in a PCR amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe, the nucleic acid to be detected and Taq enzyme. If the nucleic acid to be detected contains the target nucleic acid, both the upstream primer and the downstream primer are extended. When the extension reaches the position of the tag probe, Taq enzyme cuts off the tag sequence. The cut-off tag sequence is captured by the detection probe. The cut-off tag sequence and the detection probe form a double-stranded product by complementarity. At this time, the quencher group on the detection probe quenches the fluorophore on the tag sequence. As the amplification reaction proceeds, the number of tag probes cut off by Taq enzyme gradually increases, and the number of tag sequences captured by the detection probe increases. Finally, a double-stranded product for melting curve detection is obtained. Step 4: Perform melting curve detection on the double-stranded product obtained in Step 3 to determine whether the nucleic acid to be detected contains the target nucleic acid.

6. The method according to claim 5, wherein The length of the front part of the 5'-end tag of the 5'-end tag sequence is 4 - 30 bp, and the length of the rear part of the 5'-end tag of the 5'-end tag sequence is 3 - 5 bp.

7. The method according to claim 5, wherein The length of the 3'-end target-specific sequence is 10 - 40 bp.

8. The method according to claim 5, wherein The modification of preventing extension at the 3'-end of the tag probe is phosphate modification.

9. A method for detecting a target nucleic acid by melting curve, characterized in that, A method for detecting a target nucleic acid by melting curve detection of a double-stranded product formed by complementarity between a tag sequence extension product and a detection probe. The method includes the following steps: Step 1: Design specific upstream primer, downstream primer and tag probe for the target nucleic acid sequence. The upstream primer and downstream primer specifically bind to the target nucleic acid. The tag probe includes a 5'-end tag sequence and a 3'-end target-specific sequence. The tag sequence cannot bind to the target nucleic acid and is modified with a fluorophore. The target-specific sequence can specifically bind to the target nucleic acid. The first to fifth bases of the target-specific sequence are one or more consecutive RNA bases. The target-specific sequence is modified with a quencher group. The 3'-end of the tag probe is modified with a group that blocks extension. The quencher group on the target-specific sequence quenches the fluorophore on the tag sequence. Step 2: Design a detection probe for the 5'-end tag sequence. The detection probe is modified with at least one quencher group. There is a sequence on the detection probe that can be complementary to the tag sequence and can capture the tag sequence. Step 3: Perform amplification in a PCR amplification system containing the upstream primer, the downstream primer, the tag probe, the detection probe, the nucleic acid to be tested, RNase H enzyme, and Taq enzyme. If the nucleic acid to be tested contains the target nucleic acid, the upstream primer, the downstream primer, and the tag probe all bind to the target to be tested during annealing. The upstream primer and the downstream primer both extend. The target-specific sequence of the tag probe binds to the target nucleic acid to form a DNA / RNA hybrid strand. RNase can specifically hydrolyze the phosphodiester bond on the DNA / RNA hybrid strand to obtain the hydrolyzed tag sequence. The hydrolyzed tag sequence is captured by the detection probe and extended. The extended product of the tag sequence and the detection probe are complementary to form a double-stranded product. At this time, the quencher group on the detection probe quenches the fluorophore on the tag sequence. As the amplification reaction progresses, the tag probes hydrolyzed by RNase gradually increase, and the tag sequences captured by the detection probe and extended increase. Finally, a double-stranded product for melting curve detection is obtained. Step 4: Perform melting curve detection on the double-stranded product obtained in Step 3 to determine whether the nucleic acid to be tested contains the target nucleic acid.

10. The method according to claim 9, wherein The 4 - 30 bp at the 5'-end of the tag probe is the tag sequence.

11. The method according to claim 9, wherein The 10 - 40 bp at the 3'-end of the tag probe is the target-specific sequence.

12. The method according to claim 9, characterized in that, The modification of blocking extension at the 3'-end of the tag probe is phosphate modification.

13. A PCR kit for detecting target nucleic acid based on melting curve, characterized in that, The kit is applied in any method of Claims 1 - 12.

14. A multiplex PCR kit for detecting multiple target nucleic acids based on melting curves, characterized in that, The multiplex PCR kit detects the target nucleic acid through the melting curve of the double-stranded product formed by the complementary extension product of the tag sequence and the detection probe. The kit includes specific upstream primer, downstream primer and tag probe designed for the target nucleic acid sequence. The upstream primer and downstream primer specifically bind to the target nucleic acid. The tag probe is selected from one or more of the following three tag probes: The first labeled probe, the first labeled probe comprising a 5'-end label sequence and a 3'-end target-specific sequence, the label sequence being unable to bind to the target nucleic acid, the label sequence being modified with a fluorophore, the target-specific sequence being capable of specifically binding to the target nucleic acid, the target-specific sequence having a quenching group, the 3'-end of the labeled probe being modified with a group that blocks extension, and the quenching group on the target-specific sequence quenching the fluorophore on the label sequence; The second labeled probe, the second labeled probe comprising a 5'-end label sequence and a 3'-end target-specific sequence, the 5'-end label sequence comprising a front part and a rear part of the 5'-end label, the front part being capable of binding to the detection probe and unable to bind to the target nucleic acid, the rear part being unable to bind to the detection probe and the target nucleic acid and serving to block extension, the label sequence being modified with a fluorophore, the target-specific sequence being capable of specifically binding to the target nucleic acid, the target-specific sequence having a quenching group, the 3'-end of the labeled probe being modified with a group that blocks extension, and the quenching group on the target-specific sequence quenching the fluorophore on the label sequence; The third labeled probe, the third labeled probe comprising a 5'-end label sequence and a 3'-end target-specific sequence, the label sequence being unable to bind to the target nucleic acid, the label sequence being modified with a fluorophore, the target-specific sequence being capable of specifically binding to the target nucleic acid, the 1st to 5th bases of the target-specific sequence being one or more consecutive RNA bases, the target-specific sequence having a quenching group, the 3'-end of the labeled probe being modified with a group that blocks extension, and the quenching group on the target-specific sequence quenching the fluorophore on the label sequence; The detection probe, the detection probe being designed for the 5'-end label sequence, the detection probe being modified with a quenching group, the number of the modified quenching groups being ≥1, and the detection probe having a sequence complementary to the label sequence and being capable of capturing the label sequence; When the third labeled probe is not selected, the kit further comprises Taq enzyme; when the third labeled probe is selected, the kit simultaneously comprises Taq enzyme and RNase enzyme.

15. The kit according to claim 14, characterized in that, The labeled probe is selected from the first labeled probe and the second labeled probe.

Images