WO2022179419A1

WO2022179419A1 - Probe melting curve analysis -based multiplex nucleic acid detection method and kit

Info

Publication number: WO2022179419A1
Application number: PCT/CN2022/076520
Authority: WO
Inventors: 雷向东
Original assignee: 上海境象生物科技有限公司
Priority date: 2021-02-27
Filing date: 2022-02-16
Publication date: 2022-09-01
Also published as: CN114958988A

Abstract

The present invention relates to a probe melting curve analysis-based multiplex nucleic acid detection method. The method comprises the following steps: step A: designing an asymmetric PCR system; step B: PCR amplification; and step C: melting curve analysis. The present invention solves the significant problem that traditional Taqman technology cannot perform melting curve analysis after amplification so as to further judge whether a probe region sequence is completely consistent with a target sequence. The present invention can perform melting curve analysis after PCR amplification, can improve the detection multiplicity of fluorescence PCR, and can also perform genotyping and quantification at the same time.

Description

基于探针熔解曲线分析的多重核酸检测方法和试剂盒Multiplex nucleic acid detection method and kit based on probe melting curve analysis

技术领域technical field

本发明涉及核酸扩增和荧光PCR技术领域，具体涉及基于探针熔解曲线分析的多重核酸检测方法和试剂盒。The present invention relates to the technical field of nucleic acid amplification and fluorescent PCR, in particular to a multiple nucleic acid detection method and kit based on probe melting curve analysis.

背景技术Background technique

荧光PCR具有定量准确，使用简便，快捷，经济和PCR污染风险低的优点，是分子检测极其重要的技术。特别是Taqman技术，采用了探针，增强了检测专一性，为荧光PCR在临床的广泛使用做出了巨大贡献。然而荧光PCR也存在明显的缺点，存在技术发展的迫切需要。首先，现在的Taqman技术不能在PCR后进行熔解曲线分析，不能进一步确定探针与目标序列是否完全一致，从而不能发现很多潜在的问题。此外，荧光PCR通常一个通道检测一个靶标，检测多重度低，不适应科研特别是临床检测发展的需要。本发明公开了一种基于探针熔解曲线分析的方法，很好地解决了Taqman不能进行熔解曲线分析的问题，并且可以提升荧光PCR的检测多重度。Fluorescent PCR has the advantages of accurate quantification, easy use, fast, economical and low risk of PCR contamination, and is an extremely important technology for molecular detection. In particular, Taqman technology, which uses probes to enhance the specificity of detection, has made a great contribution to the widespread use of fluorescent PCR in clinical practice. However, fluorescent PCR also has obvious shortcomings, and there is an urgent need for technical development. First, the current Taqman technology cannot perform melting curve analysis after PCR, and cannot further determine whether the probe is completely consistent with the target sequence, so many potential problems cannot be found. In addition, fluorescent PCR usually detects one target in one channel, and the detection multiple is low, which is not suitable for the needs of scientific research, especially the development of clinical detection. The invention discloses a method based on probe melting curve analysis, which well solves the problem that Taqman cannot perform melting curve analysis, and can improve the detection multiplicity of fluorescent PCR.

Taqman技术采用自淬灭荧光探针和具有5’->3’外切酶活力的DNA聚合酶实时检测PCR扩增过程。自淬灭探针5’标记报告荧光基团，3’标记荧光淬灭基团。在游离存在时，自淬灭探针由于淬灭基团对报告基团的淬灭作用，本底荧光较弱，而在PCR引物延伸时，通过DNA聚合酶的5’->3’外切酶的作用，标记在5’端的报告基团被酶切，使得报告基团与3’端的淬灭基团分离，报告基团发出强的荧光。此过程在PCR扩增每个循环重复，PCR过程得到实时监控。Taqman技术实现了传统终端法PCR到实时荧光PCR的伟大转变，并通过Ct(threshhold cycle)值对目标序列进行准确定量的革命性的进步。同时由于Taqman技术不需要在PCR后再将PCR产物取出进行后PCR分析，从根本上解决了PCR检测的致命的PCR污染痛点问题，极大地推动了荧光PCR在临床上的应用，创造了分子检测的辉煌。Taqman technology uses self-quenching fluorescent probes and DNA polymerases with 5'->3' exonuclease activity to detect the PCR amplification process in real time. The self-quenching probe is labeled with a reporter fluorophore at 5' and a fluorescent quencher at 3'. In the free presence of self-quenching probes, the background fluorescence of the self-quenching probe is weak due to the quenching effect of the quenching group on the reporter group, and when the PCR primer is extended, the 5'->3' excision of the DNA polymerase occurs. Due to the action of the enzyme, the reporter group labeled at the 5' end is cleaved by the enzyme, so that the reporter group is separated from the quencher group at the 3' end, and the reporter group emits strong fluorescence. This process is repeated for each cycle of PCR amplification, and the PCR process is monitored in real time. Taqman technology realizes a great transformation from traditional terminal PCR to real-time fluorescent PCR, and makes a revolutionary progress in accurate quantification of target sequences through Ct (threshhold cycle) value. At the same time, since Taqman technology does not require PCR products to be taken out for post-PCR analysis, it fundamentally solves the fatal PCR contamination pain point of PCR detection, greatly promotes the clinical application of fluorescent PCR, and creates molecular detection. of brilliance.

然而，Taqman技术也有其固有缺陷，那就是Taqman技术不能像荧光染料法定量PCR那样，对PCR产物进行熔解曲线分析，因为探针在扩增过程被降解了。即使有少量完整的探针留下，也因为PCR过程中大量被降解的探针，具有很强的荧光背景，而掩盖了熔解曲线分析时微小的荧光信号的变化，使得熔解曲线分析没有意义。However, Taqman technology also has its inherent defects, that is, Taqman technology cannot perform melting curve analysis of PCR products like fluorescent dye quantitative PCR, because the probe is degraded during the amplification process. Even if there is a small amount of intact probe left, because a large number of degraded probes in the PCR process have a strong fluorescent background, which masks the slight changes in the fluorescence signal during the melting curve analysis, making the melting curve analysis meaningless.

正如Taqman技术本身所声称的一样，Taqman技术已经使用探针对PCR产物进行鉴别，大大提升了专一性，没有必要对PCR产物进行熔解曲线分析。这是本领域技术人员公知常识。As the Taqman technology itself claims, the Taqman technology already uses probes to identify PCR products, greatly improving specificity and eliminating the need for melting curve analysis of PCR products. This is common knowledge of those skilled in the art.

发明人在该技术应用过程中发现，如果对Taqman技术进行发展，使其能够对PCR产物进行熔解曲线分析，将解决Taqman技术的一些技术问题，使其具有更好的应用前景，以下列举几种情况说明：During the application of this technology, the inventor found that if Taqman technology is developed to enable melting curve analysis of PCR products, some technical problems of Taqman technology will be solved and it will have better application prospects. The following lists several Situation Description:

(1)产生假阳性结果。当样本存在序列相似的目标序列(如同源基因或同一家族不同成员基因)时，PCR引物和探针很有可能在目标序列和非目标序列相差很小，因此PCR引物会扩增非目标序列，探针也可以与非目标序列结合而在扩增中被降解，只是效率较差而已。结果是产生假阳性结果。(1) Produces false positive results. When there are target sequences with similar sequences in the sample (such as homologous genes or genes of different members of the same family), the PCR primers and probes are likely to have little difference between the target sequence and the non-target sequence, so the PCR primers will amplify the non-target sequence, Probes can also bind to non-target sequences and be degraded during amplification, albeit less efficiently. The result is a false positive result.

解决途径：如果PCR后可以进行探针熔解曲线分析，根据测到的探针熔解温度，可以判断探针检测区域是否为目标序列。如果不是目标序列，测到的探针Tm值要小于探针与目标序列的双链的Tm值。Solution: If the probe melting curve analysis can be performed after PCR, it can be determined whether the probe detection region is the target sequence according to the measured probe melting temperature. If it is not the target sequence, the measured Tm value of the probe should be smaller than the Tm value of the double strand between the probe and the target sequence.

(2)影响弱阳性样本的判断。基于Taqman技术的检测经常会遇到扩增曲线在扩增过程快结束时出现翘尾的情况，很难分清究竟是弱阳性样本还是由于非特异性扩增所导致。通常的做法是被迫设定较小的Ct值作为阳性判断标准，结果是降低了检测灵敏度，影响了弱阳性样本的检出。(2) It affects the judgment of weakly positive samples. The detection based on Taqman technology often encounters the situation that the amplification curve is tilted at the end of the amplification process, and it is difficult to distinguish whether it is a weak positive sample or a non-specific amplification. The usual practice is to be forced to set a smaller Ct value as the positive judgment standard, which reduces the detection sensitivity and affects the detection of weakly positive samples.

解决途径：如果PCR后可以进行探针熔解曲线分析，根据测到的探针熔解温度，可以判断是弱阳性样本还是阴性样本。如果测到的探针Tm值正确，样本是弱阳性样本。Solution: If the probe melting curve analysis can be performed after PCR, it can be judged whether it is a weak positive sample or a negative sample according to the measured melting temperature of the probe. If the measured probe Tm value is correct, the sample is a weak positive sample.

(3)产生假阴性结果。当目标序列在探针区域存在突变，且突变导致探针与目标序列结合的Tm大幅下降，探针不能在PCR扩增过程与目标序列结合而被降解，结果是产生假阴性。(3) produce false negative results. When the target sequence has a mutation in the probe region, and the mutation causes the Tm of the probe to bind to the target sequence to be greatly reduced, the probe cannot be combined with the target sequence during the PCR amplification process and is degraded, resulting in a false negative.

解决途径：如果PCR后可以进行探针熔解曲线分析，根据测到的探针熔解温度，可以判断样本是否真是阴性样本。如果熔解曲线呈现显著的熔解曲线峰，且Tm值小于正确的Tm值，说明探针区域很有可能发生了突变，需要测序核实，不能简单报为阴性结果。Solution: If the probe melting curve analysis can be performed after PCR, it can be judged whether the sample is really a negative sample according to the measured melting temperature of the probe. If the melting curve shows a significant melting curve peak, and the Tm value is less than the correct Tm value, it indicates that the probe region is likely to have a mutation, which needs to be verified by sequencing, and cannot be simply reported as a negative result.

(4)导致定量错误。当目标序列在探针区域存在突变时，如果突变没有造成Tm值的巨大改变，探针依然可以检测到目标序列，但效率降低了。宏观表现是同一浓度的样本，探针区域发生突变的样本比没有发生突变的样本Ct值大，结果是样本浓度被低估了。(4) lead to quantitative errors. When the target sequence has a mutation in the probe region, if the mutation does not cause a huge change in the Tm value, the probe can still detect the target sequence, but the efficiency is reduced. The macroscopic expression is that the samples with the same concentration, the samples with mutation in the probe region have a larger Ct value than the samples without mutation, and the result is that the sample concentration is underestimated.

解决途径：如果PCR后可以进行探针熔解曲线分析，根据测到的探针熔解温度，可以判断定量是否低估。如果测到的Tm值小于正确的Tm值，说明探针区域很有可能发生了突变，此时定量结果会偏低。Solution: If the probe melting curve analysis can be performed after PCR, it can be judged whether the quantification is underestimated according to the measured probe melting temperature. If the measured Tm value is less than the correct Tm value, it indicates that the probe region is likely to have a mutation, and the quantitative result will be low.

综上所述，以为Taqman技术采用了探针，检测到的结果就一定是正确的，这只是一种想当然。如果应用Taqman技术也能在PCR扩增后进行熔解曲线分析，将可以帮助判断包括以上列举的多种情况。长久以来，一直有各种努力，希望Taqman技术也能在扩增后进行熔解曲线分析，但至今未有满意的通用解决方法。To sum up, it is only a matter of course to think that the Taqman technology uses probes and the detected results must be correct. If Taqman technology can also be used for melting curve analysis after PCR amplification, it will help to judge the various situations listed above. For a long time, various efforts have been made to hope that Taqman technology can also perform melting curve analysis after amplification, but there is no satisfactory general solution so far.

现有荧光PCR技术的另一个重要缺陷是多重度低，一般一个通道检测一个靶标，常见的4通道荧光PCR仪只能检测4个靶标。采用多重探针熔解曲线法，理论上可以提高检测多重度。但是，多重探针熔解曲线分析技术开发难度大，体系不稳定，至今没有成熟和公开的技术，本发明在大量的研发中发现了一种可控制可重复并普遍适应的多重探针熔解曲线方法。该技术不仅可以用于定性检测，还可以用于定量检测。Another important defect of the existing fluorescent PCR technology is the low multiplicity. Generally, one channel detects one target, and the common 4-channel fluorescent PCR instrument can only detect four targets. Using the multiple probe melting curve method can theoretically improve the detection multiplex. However, the development of multiple probe melting curve analysis technology is difficult, the system is unstable, and there is no mature and disclosed technology so far. The present invention has found a controllable, repeatable and universally applicable multiple probe melting curve method in a lot of research and development. . This technique can be used not only for qualitative detection but also for quantitative detection.

PCR-探针熔解曲线分析产品研发难度大，体系不稳定，还有一个重要原因是检测探针与检测区域的杂交面临两个竞争：一个是PCR产物单链形成分子内高级结构，从而阻止检测探针与检测区域的结合：情况轻微者，表现是熔解曲线峰峰高很低，影响结果判断；情况严重者，完全没有熔解曲线峰，导致假阴性。从理论分析，任何一定长度的DNA单链都有形成不同程度的稳定结构的趋势。由于DNA单链形成高级结构是分子内反应，而探针与PCR产物单链形成双链是分子间反应，前者具有明显的反应动力学的优势。要克服这种不利影响，提高探针的熔解温度是最有效和最容易想到的办法，但是，探针的Tm值受检测序列的制约，比如对基因多样性或基因突变的检测，目标序列位置已大致确定，没有很大选择余地；同时要区分野生型和突变型，更进一步限制了Tm值的设计选择空间。另一个竞争是PCR产物双链的结合。这种结合虽然也是分子间反应，但PCR产物的Tm一般都大大高于检测探针与检测序列形成双链的Tm，因此前者具有优势。本发明公开了使用辅助序列解决这个问题的方法。PCR-probe melting curve analysis products are difficult to develop and the system is unstable. Another important reason is that the hybridization between the detection probe and the detection region faces two competitions: one is that the single strand of the PCR product forms an intramolecular high-level structure, which prevents detection The combination of the probe and the detection area: in mild cases, the peak height of the melting curve is very low, which affects the judgment of the results; in severe cases, there is no melting curve peak at all, resulting in false negatives. From a theoretical analysis, DNA single strands of any certain length have a tendency to form stable structures of varying degrees. Since the formation of higher-order structures by DNA single strands is an intramolecular reaction, and the formation of double strands between probes and PCR product single strands is an intermolecular reaction, the former has the advantage of obvious reaction kinetics. To overcome this adverse effect, increasing the melting temperature of the probe is the most effective and easiest way to think. However, the Tm value of the probe is restricted by the detection sequence, such as the detection of gene diversity or gene mutation, the position of the target sequence It has been roughly determined that there is not much room for choice; at the same time, the need to distinguish between wild type and mutant type further limits the design choice space of Tm value. Another competition is the binding of the PCR product duplexes. Although this combination is also an intermolecular reaction, the Tm of the PCR product is generally much higher than the Tm of the detection probe and the detection sequence to form a double strand, so the former has an advantage. The present invention discloses a method for solving this problem using helper sequences.

发明内容SUMMARY OF THE INVENTION

鉴于上述现有技术中存在的问题，本申请发明人进行了深入研究，在多重熔解曲线技术研究中意外的发现，基于Taqman技术，能在扩增后进行熔解曲线分析这个问题可以巧妙而有效地解决：In view of the problems existing in the above-mentioned prior art, the inventor of the present application has conducted in-depth research, and unexpectedly found in the research of multiple melting curve technology, based on Taqman technology, the problem that melting curve analysis can be carried out after amplification can be skillfully and effectively solve:

将PCR正向引物和反向引物设计成Tm值相差几度，在PCR扩增体系中低Tm值的引物(简称低温引物)浓度高于高Tm值的引物(简称高温引物)浓度，而且探针和高温引物与目标序列PCR产物的同一条链杂交(即探针设计在高温引物的目标链上)，我们惊奇的发现PCR扩增后进行熔解曲线分析，我们得到了熔解曲线峰，和正确、稳定的探针Tm值。The PCR forward primer and reverse primer are designed so that the Tm values differ by a few degrees. In the PCR amplification system, the concentration of primers with low Tm values (referred to as low temperature primers) is higher than that of primers with high Tm values (referred to as high temperature primers), and the The needle and the high temperature primer hybridize to the same strand of the PCR product of the target sequence (that is, the probe is designed on the target strand of the high temperature primer), we were surprised to find that the melting curve analysis was performed after PCR amplification. , Stable probe Tm value.

在常规的定量PCR可以接受的引物、探针浓度范围内设计PCR体系，通常在PCR体系中的终浓度为0.01-10umol/L,引物、探针Tm值经过公式或软件(例如Tm calculator、OligoAnalyzer Tool，Primer3)进行计算并通过有限次的实验，获得符合要求的Tm值，一个简单的Tm值计算公式为：The PCR system is designed within the acceptable concentration range of primers and probes for conventional quantitative PCR. Usually, the final concentration in the PCR system is 0.01-10 umol/L. The Tm values of primers and probes are determined by formulas or software (such as Tm calculator, OligoAnalyzer Tool, Primer3) to calculate and obtain the required Tm value through a limited number of experiments. A simple Tm value calculation formula is:

Tm＝4(G+C)+2(A+T)℃。Tm=4(G+C)+2(A+T)°C.

探针和高温引物与目标序列PCR产物的同一条链杂交，达到的效果是PCR扩增中只有当高温引物被用于合成PCR产物时，探针才会被降解。由于探针浓度高于高温引物浓度，这种设计注定在PCR扩增结束后依然有一定浓度的完整的探针存在，这些完整的探针在熔解曲线分析时发挥了作用。而将PCR正向引物和反向引物设计成相差几度，解决了由于高温引物(低浓度)用量少于低温引物(高浓度)时PCR扩增的效率降低的问题。The probe and the high-temperature primer hybridize to the same strand of the PCR product of the target sequence, and the effect is that the probe will be degraded only when the high-temperature primer is used to synthesize the PCR product in the PCR amplification. Since the probe concentration is higher than the high-temperature primer concentration, this design is destined to have a certain concentration of intact probes after PCR amplification, and these intact probes play a role in melting curve analysis. The PCR forward primer and reverse primer are designed to differ by a few degrees, which solves the problem that the efficiency of PCR amplification decreases when the amount of high temperature primer (low concentration) is less than that of low temperature primer (high concentration).

应用本发明公开的技术，发明人测试了多种基于Taqman技术的检测体系，都可以进行熔解曲线分析，并得到了有意义的熔解曲线。因此本发明解决了传统的Taqman技术不能在扩增后进行熔解曲线分析以进一步判断探针区域序列是否与目标序列完全一致的重要问题，是对Taqman技术的重要发展。Using the technology disclosed in the present invention, the inventors have tested a variety of detection systems based on Taqman technology, all of which can perform melting curve analysis and obtain meaningful melting curves. Therefore, the present invention solves the important problem that the traditional Taqman technology cannot perform melting curve analysis after amplification to further determine whether the probe region sequence is completely consistent with the target sequence, and is an important development of the Taqman technology.

本发明的基于探针熔解曲线分析的多重核酸检测方法包括以下步骤：The multiple nucleic acid detection method based on probe melting curve analysis of the present invention comprises the following steps:

A)不对称PCR体系设计；A) Asymmetric PCR system design;

B)PCR扩增；B) PCR amplification;

C)熔解曲线分析。C) Melting curve analysis.

步骤A.不对称PCR体系设计。Step A. Asymmetric PCR system design.

优选，所述步骤A的不对称PCR体系设计包括：Preferably, the asymmetric PCR system design of the step A includes:

A1.针对每个目标核酸序列设计正向和反向PCR引物，每个目标序列设计至少一对引物，针对多个目标核酸序列设计简并引物，或设计多条引物；和A1. Design forward and reverse PCR primers for each target nucleic acid sequence, design at least one pair of primers for each target sequence, design degenerate primers for multiple target nucleic acid sequences, or design multiple primers; and

A2.对每个目标核酸序列设计一条探针，探针设计位置位于正向和反向引物之间；A2. Design a probe for each target nucleic acid sequence, and the probe design position is located between the forward and reverse primers;

其中A1中正向和反向引物的Tm值不同，引物在PCR扩增体系中低温引物浓度高于高温引物；A2中探针和高温引物与目标序列的同一条链杂交。The Tm values of the forward and reverse primers in A1 are different, and the primers in the PCR amplification system have a higher concentration of low-temperature primers than high-temperature primers; in A2, the probe and high-temperature primers hybridize to the same strand of the target sequence.

优选，所述步骤A的不对称PCR体系设计进一步包括：Preferably, the asymmetric PCR system design of the step A further comprises:

A3.DNA聚合酶、dNTP、镁离子浓度设计，例如，DNA聚合酶的浓度为0.05-10U/体系，镁离子的浓度为1-6mM，和，dNTP(含U或不含U)的浓度为0.01-10mM。A3. DNA polymerase, dNTP, magnesium ion concentration design, for example, the concentration of DNA polymerase is 0.05-10 U/system, the concentration of magnesium ion is 1-6 mM, and, the concentration of dNTP (with or without U) is 0.01-10mM.

更具体地说，所述步骤A的不对称PCR体系设计包括：More specifically, the asymmetric PCR system design of the step A includes:

A1.引物设计和不对称引物浓度设计：A1. Primer design and asymmetric primer concentration design:

在步骤A)的引物设计中，将PCR正向引物和反向引物设计成Tm值相差0.1-15℃(例如0.3，0.5，0.7，0.8，0.9，1，2，3，4，5，6，7，8，9，11，12，13或14℃),优选的相差3-10℃，Tm值低的引物浓度(按摩尔浓度计)高于Tm值高的引物浓度，如低Tm引物浓度与高Tm引物浓度的比值为1.05至30(例如，1.2，1.3，1.5，1.8，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20，21，22，23，24，25，26，27，28，29)，优选的，1.3至25或1.3至5，更优选的，2至15，即，(2-15)：1。In the primer design of step A), the PCR forward primer and reverse primer are designed so that the Tm values differ by 0.1-15°C (for example, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 11, 12, 13 or 14°C), the preferred difference is 3-10°C, the concentration of primers with low Tm value (in molar concentration) is higher than that of primers with high Tm value, such as low Tm primers The ratio of concentration to high Tm primer concentration is 1.05 to 30 (e.g., 1.2, 1.3, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29), preferably, 1.3 to 25 or 1.3 to 5, more preferably, 2 to 15, i.e. , (2-15): 1.

在常规的定量PCR可以接受的引物、探针浓度范围内设计PCR体系，通常在PCR体系中的终浓度为0.01-10umol/L(例如，0.02，0.05，0.07，0.08，0.09，0.1，0.2，0.3，0.4，0.5，0.6，0.7，0.8，1，1.2，1.5，1.8，2，2.5，3，3.5，4，4.5，5，5.5，6，7，8，9umol/L)；引物、探针Tm值经过相关软件进行计算并通过有限次的实验调整，获得符合要求的Tm值。The PCR system is designed within the acceptable concentration range of primers and probes for conventional quantitative PCR, usually the final concentration in the PCR system is 0.01-10 umol/L (for example, 0.02, 0.05, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9umol/L); primers, probes The needle Tm value is calculated by relevant software and adjusted through a limited number of experiments to obtain a Tm value that meets the requirements.

A2.探针设计：A2. Probe Design:

在步骤A)的探针设计中，如果检测完全不同的目标序列，对每个目标核酸序列设计一条探针；如果用于基因突变或基因多样性，如单核苷酸多样性SNP或少数几个碱基(≤3个)的缺失或***，可以只设计一条探针，设计的探针可同时识别野生型目标序列和突变的目标序列；对于碱基差别≥3个的情况，需要设计多条探针。探针位于正向和反向引物之间，且探针和Tm值高的引物与目标序列的同一条链杂交。In the probe design of step A), if detecting completely different target sequences, design a probe for each target nucleic acid sequence; if it is used for gene mutation or gene diversity, such as single nucleotide diversity SNP or a few For the deletion or insertion of 3 bases (≤3), only one probe can be designed, and the designed probe can recognize both the wild-type target sequence and the mutated target sequence; for the case of base differences ≥3, it is necessary to design more than one probe. strip probe. The probe is located between the forward and reverse primers, and the probe and the primer with a high Tm value hybridize to the same strand of the target sequence.

同一荧光通道内，针对需要区分的不同目标序列，探针Tm值不同，另外，不需要区分目标序列的，探针Tm值可以相同(或不同)，不同荧光通道的探针Tm值可以相同(或不同)；Tm范围在40-80℃(例如，45，50，55，60，65，70，75℃)。相邻的Tm值的差值是在2-35℃的范围内(例如，3，4，5，6，7，8，9，10，11，12，13，15，18，20，22，25，27，30或33℃)，优选的，在2.5-16℃，更优选的，在3-12℃。In the same fluorescence channel, for different target sequences that need to be distinguished, the probe Tm values are different. In addition, if there is no need to distinguish the target sequences, the probe Tm values can be the same (or different), and the probe Tm values of different fluorescence channels can be the same ( or different); Tm ranges from 40-80°C (eg, 45, 50, 55, 60, 65, 70, 75°C). The difference between adjacent Tm values is in the range of 2-35°C (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 22, 25, 27, 30 or 33°C), preferably, at 2.5-16°C, more preferably, at 3-12°C.

当用于检测基因突变或基因多样性，如单核苷酸多样性SNP或少数几个碱基(≤3个)的缺失或***时，可以只设计一条探针，设计的探针可同时识别野生型目标序列和突变的目标序列；两者Tm值相差2-30℃(例如，2.5，3，3.5，4，4.5，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20，22，25，27或28℃)，优选的，在2.5-15℃，更优选的，在3-12℃。When used to detect gene mutation or gene diversity, such as single nucleotide diversity SNP or deletion or insertion of a few bases (≤3), only one probe can be designed, and the designed probe can recognize at the same time Wild-type target sequence and mutated target sequence; Tm values differ by 2-30°C (eg, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27 or 28°C), preferably, at 2.5-15°C, more preferably, at 3-12°C.

当用于检测基因突变或基因多样性，野生型序列与突变型序列相差较大时，如达到或超过3个碱基差别时，可以为野生型序列和突变序列分别设计探针，分别设计的探针Tm值相差2-30℃(例如，2.5，3，3.5，4，4.5，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20，22，25，27或28℃)，优选的，在2.5-15℃，更优选的，在3-12℃。探针的Tm设计与上述引物的Tm值设计方法相同。When it is used to detect gene mutation or gene diversity, when the difference between the wild-type sequence and the mutant-type sequence is large, such as when the difference between the wild-type sequence and the mutant-type sequence reaches or exceeds 3 bases, probes can be designed separately for the wild-type sequence and the mutant sequence. Probe Tm values differ by 2-30°C (eg, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27 or 28°C), preferably, at 2.5-15°C, more preferably, at 3-12°C. The Tm design of the probe is the same as the design method of the Tm value of the above-mentioned primers.

探针标记有荧光发色基团，如FAM、ROX、VIC、CY5、HEX、Texas Red、TET、等，和荧光淬灭基因，荧光发色基团和淬灭基团可以标记在探针的两端或非两端位置，例如5’荧光标记，3’淬灭基团；或5’淬灭基团，3’荧光标记。Probes are labeled with fluorescent chromophores, such as FAM, ROX, VIC, CY5, HEX, Texas Red, TET, etc., and fluorescent quenching genes. The fluorescent chromophores and quenching groups can be labeled on the probe. Terminal or non-terminal positions, such as 5' fluorescent label, 3' quencher group; or 5' quencher group, 3' fluorescent label.

A3.DNA聚合酶、dNTP、镁离子浓度设计；A3. DNA polymerase, dNTP, magnesium ion concentration design;

按照本领域技术人员公知常识的PCR扩增经验进行上述组分的浓度设计，例如：DNA聚合酶：0.05-10U/体系(例如0.1，0.2，0.5，0.8，1，1.5，2，3，4，5，6，7，8或9U)；镁离子:1-6mM(例如2，3，4或5mM)，dNTP(含U或不含U)：0.01-10mM(例如0.05，0.1，0.2，0.5，0.8，1，1.5，2，3，4，5，6，7，8或9mM)，也可以使用市售的预混PCR MasterMix，例如境象生物货号Ace104，楚天生物货号Ctb104，TAKARA货号RR390，天根货号FP206等等。Design the concentration of the above components according to the PCR amplification experience known to those skilled in the art, for example: DNA polymerase: 0.05-10U/system (for example, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 3, 4 , 5, 6, 7, 8 or 9U); magnesium ions: 1-6 mM (

eg

2, 3, 4 or 5 mM), dNTPs (with or without U): 0.01-10 mM (eg 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 3, 4, 5, 6, 7, 8 or 9mM), you can also use a commercially available pre-mixed PCR MasterMix, such as Ace104, Chutian Bio, Ctb104, TAKARA Item No. RR390, Tiangen Item No. FP206, etc.

进一步地，步骤A3中，用于PCR扩增的DNA聚合酶是有5’->3’外切酶活力的聚合酶(基于Taqman技术，有经典的S型扩增曲线，还可进行熔解曲线分析)，或无5’->3’外切酶活力的聚合酶(非Taqman技术，有熔解曲线，也能有经典的S型扩增曲线，可用于定量)，或两者的混合物(比采用无5’->3’外切酶活力的聚合酶有更高的扩增信号)；其中，无5’->3’外切酶活力的聚合酶：有5’->3’外切酶活力的聚合酶二者的活性比可以是1:0.005～200(优选，1:0.007～190，优选1:0.01～180，优选1:0.05～170，优选1:0.08～160，优选1:0.1～150，优选1:0.12～140，优选1:0.15～130，优选1:0.18～120，例如1:0.2，1:0.4，1:0.5，1:0.7，1:0.8，1:1，1:2，1:3，1:5，1:8，1:10，1:15，1:20，1:30，1:40，1:50，1:70，1:80，或1:100)，优选约5:1。Further, in step A3, the DNA polymerase used for PCR amplification is a polymerase with 5'->3' exonuclease activity (based on Taqman technology, there is a classic S-shaped amplification curve, and a melting curve can also be performed. analysis), or a polymerase without 5'->3' exonuclease activity (non-Taqman technology, with melting curve, can also have classical sigmoid amplification curve, which can be used for quantification), or a mixture of the two (ratio Using a polymerase without 5'->3' exonuclease activity has a higher amplification signal); among them, a polymerase without 5'->3' exonuclease activity: with 5'->3' exonuclease The activity ratio of the two polymerases of the enzymatic activity can be 1:0.005～200 (preferably, 1:0.007～190, preferably 1:0.01～180, preferably 1:0.05～170, preferably 1:0.08～160, preferably 1:0. 0.1～150, preferably 1:0.12～140, preferably 1:0.15～130, preferably 1:0.18～120, for example 1:0.2, 1:0.4, 1:0.5, 1:0.7, 1:0.8, 1:1, 1:2, 1:3, 1:5, 1:8, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:70, 1:80, or 1 :100), preferably about 5:1.

步骤B.PCR扩增Step B. PCR Amplification

按照设计好的PCR体系进行PCR反应配制，按本领域常规的方法进行PCR扩增。例如常规的三步法(变性-退火-延伸)或两步法(变性-退火延伸)PCR扩增。The PCR reaction preparation is carried out according to the designed PCR system, and the PCR amplification is carried out according to the conventional method in the art. For example conventional three-step (denaturation-annealing-extension) or two-step (denaturation-annealing-extension) PCR amplification.

步骤C.熔解曲线分析步骤Step C. Melting Curve Analysis Step

一般，步骤C中的熔解曲线分析步骤包括：常规的熔解曲线分析步骤，例如，将PCR条件(或PCR体系)降温，升温，然后再降温。Generally, the melting curve analysis step in step C includes: a conventional melting curve analysis step, for example, cooling the PCR conditions (or PCR system), raising the temperature, and then lowering the temperature again.

优选，步骤C中的熔解曲线分析步骤包括：Preferably, the melting curve analysis step in step C comprises:

C1.复性：一个缓慢的从高温到低温的DNA从单链形成双链的复性步骤，C1. Refolding: a slow annealing step from high temperature to low temperature DNA from single strand to double strand,

C2.熔解曲线分析步骤：常规的熔解曲线分析步骤，例如，将PCR条件(或PCR体系)降温，升温，然后再降温。C2. Melting curve analysis step: a conventional melting curve analysis step, for example, cooling the PCR conditions (or PCR system), raising the temperature, and then lowering the temperature again.

优选，所述步骤C包括：Preferably, the step C includes:

C1.复性：在PCR扩增步骤结束后在进行从低温到高温的熔解曲线分析前，优选的，包括一个缓慢的从高温到低温的DNA从单链形成双链的复性步骤，降温速度低于8℃/秒，优选地低于7℃/秒，优选地低于5℃/秒，优选的低于3℃/秒，更优选的低于2℃/秒。或者，分阶段降温，例如从94℃至80℃，进行常规快速降温，然后，从80℃至45℃进行缓慢降温(例如降温速度为1-20℃/分钟，如2、3、4、5、6、7、8、9、10、12、15或18℃/分钟)。C1. Refolding: after the PCR amplification step, before performing the melting curve analysis from low temperature to high temperature, it is preferable to include a slow renaturation step of DNA from high temperature to low temperature to form double strands from single strands, and the cooling rate is Below 8°C/sec, preferably below 7°C/sec, preferably below 5°C/sec, preferably below 3°C/sec, more preferably below 2°C/sec. Or, cooling in stages, for example, from 94°C to 80°C, perform conventional rapid cooling, and then perform slow cooling from 80°C to 45°C (for example, the cooling rate is 1-20°C/min, such as 2, 3, 4, 5 , 6, 7, 8, 9, 10, 12, 15 or 18°C/min).

C2.熔解曲线分析步骤：C2. Melting curve analysis steps:

常规的熔解曲线分析步骤，例如，将PCR条件(或PCR体系)降温到40℃左右(例如37-45℃)，再升温到80-95℃，再降温到40℃左右(例如37-45℃)，具体降温速度和时间根据PCR仪的说明书进行常规设定。Conventional melting curve analysis steps, for example, cooling the PCR conditions (or PCR system) to about 40°C (eg 37-45°C), then heating to 80-95°C, and then cooling to about 40°C (eg 37-45°C) ), and the specific cooling rate and time are routinely set according to the instructions of the PCR instrument.

本发明的方法在一个反应中检测多个目标核酸序列，利用特别设计的引物通过PCR扩增多个目标核酸序列，利用与目标序列形成双链后具有不同熔解温度(Tm)的多条荧光标记探针对PCR产物进行熔解曲线分析，根据荧光标记和Tm值对结果进行判读，实现一管反应对多个目标序列的多重检测或基因分型；同时利用探针实时监测扩增过程，并获取扩增的Ct值，用于目标序列的定量评估。The method of the present invention detects multiple target nucleic acid sequences in one reaction, uses specially designed primers to amplify multiple target nucleic acid sequences by PCR, uses multiple fluorescent labels that have different melting temperatures (Tm) after forming double strands with the target sequences The probe performs melting curve analysis on the PCR product, and interprets the results according to the fluorescent label and Tm value, realizing multiple detection or genotyping of multiple target sequences in one tube reaction; at the same time, the probe is used to monitor the amplification process in real time, and obtain Amplified Ct values for quantitative assessment of target sequences.

本发明进一步提供一种基于探针熔解曲线分析的多重核酸检测试剂盒，该试剂盒包括：The present invention further provides a multiplex nucleic acid detection kit based on probe melting curve analysis, the kit comprising:

针对每个目标核酸序列设计的正向和反向PCR引物，每个目标序列设计至少一对引物，针对多个目标核酸序列可以设计简并引物，或设计多条引物；Forward and reverse PCR primers designed for each target nucleic acid sequence, at least one pair of primers designed for each target sequence, degenerate primers can be designed for multiple target nucleic acid sequences, or multiple primers can be designed;

针对每个目标核酸序列设计的一条探针，探针设计位置位于正向和反向引物之间，针对不同目标序列的探针Tm值不同；A probe designed for each target nucleic acid sequence, the probe design position is located between the forward and reverse primers, and the probe Tm values for different target sequences are different;

以及用于PCR扩增的DNA聚合酶和其它PCR组分如dNTP、镁离子；and DNA polymerase and other PCR components such as dNTPs, magnesium ions for PCR amplification;

其中正向和反向引物的Tm值不同，引物在PCR扩增体系中Tm值低的引物浓度高于Tm值高的引物；探针和Tm值高的引物与目标序列的同一条链杂交。The Tm values of the forward and reverse primers are different, and the primers with low Tm values in the PCR amplification system have a higher concentration than those with high Tm values; the probe and the primers with high Tm values hybridize to the same strand of the target sequence.

优选地，高温和低温引物的Tm值相差0.1-15℃；低Tm引物浓度与高Tm引物浓度的比值为1.05至30；A2步骤中，探针Tm范围在40-80℃；相邻的Tm值的差值在2-35℃。Preferably, the Tm values of the high temperature and low temperature primers differ by 0.1-15°C; the ratio of the low Tm primer concentration to the high Tm primer concentration is 1.05 to 30; in step A2, the probe Tm range is 40-80°C; adjacent Tm The difference in values is between 2-35°C.

优选地，DNA聚合酶为无5’->3’外切酶活力的聚合酶，或有5’->3’外切酶活力的聚合酶，或两者的混合物。Preferably, the DNA polymerase is a polymerase without 5'->3' exonuclease activity, or a polymerase with 5'->3' exonuclease activity, or a mixture of the two.

本发明的方法优选进一步包括在步骤B中使用辅助序列，所述辅助序列为长度5-200个碱基的核酸序列，辅助序列与检测探针在与PCR产物杂交区域不重叠，优选地，所述辅助序列3’端进行修饰以阻止其被延长。优选，所述辅助序列为长度6-195个，优选7-190个，优选8-180个，优选9-170个碱基的核酸序列，例如碱基的数量为10，20，30，40，50，60，70，80，90，100，110，120，130，140，150或160个。The method of the present invention preferably further comprises using an auxiliary sequence in step B, the auxiliary sequence is a nucleic acid sequence with a length of 5-200 bases, and the auxiliary sequence and the detection probe do not overlap in the hybridization region with the PCR product. The 3' end of the helper sequence is modified to prevent it from being elongated. Preferably, the auxiliary sequence is a nucleic acid sequence with a length of 6-195, preferably 7-190, preferably 8-180, preferably 9-170 bases, for example, the number of bases is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 pieces.

本发明还公开了一种提高PCR熔解曲线分析灵敏度的方法，包括步骤A：引物、探针、辅助序列设计；步骤B：PCR扩增；步骤C：熔解曲线分析；特征在于使用辅助序列。辅助序列可以有一个或多个，所述辅助序列为长度5-200个碱基的核酸序列，采用多条辅助序列时，可与PCR产物的任何一条DNA链互补；辅助序列不能与PCR的引物、探针、其它辅助序列互补；辅助序列与引物或检测探针在与PCR产物杂交区域不重叠；为避免其被当做引物延伸，需对其3’端进行化学修饰。The invention also discloses a method for improving the sensitivity of PCR melting curve analysis, including step A: primer, probe, auxiliary sequence design; step B: PCR amplification; step C: melting curve analysis; characterized in that the auxiliary sequence is used. There can be one or more auxiliary sequences. The auxiliary sequences are nucleic acid sequences with a length of 5-200 bases. When multiple auxiliary sequences are used, they can be complementary to any DNA strand of the PCR product; the auxiliary sequences cannot be used with PCR primers. , probe, and other auxiliary sequences are complementary; the auxiliary sequence does not overlap with the primer or detection probe in the hybridization region with the PCR product; in order to prevent it from being used as a primer extension, its 3' end needs to be chemically modified.

辅助序列可以有几个作用：Auxiliary sequences can serve several purposes:

1.与检测探针协同杂交：一条或多条辅助序列与检测探针一起，与模板杂交，能够增强检测探针与模板的结合能力。1. Synergistic hybridization with the detection probe: one or more auxiliary sequences are hybridized with the template together with the detection probe, which can enhance the binding ability of the detection probe and the template.

2.破坏PCR产物高级结构：例如与容易产生高级结构的部位结合，从而阻止PCR产物内部产生高级结构。2. Destruction of the higher-order structure of the PCR product: For example, it is combined with the site where the higher-order structure is easily generated, thereby preventing the generation of the higher-order structure inside the PCR product.

3.阻止PCR产物两条链自身结合，从而增加检测探针与检测序列的结合。3. Prevent the two chains of PCR products from binding themselves, thereby increasing the binding of the detection probe to the detection sequence.

另外，本发明的提高PCR探针熔解曲线分析灵敏度的方法包括以下步骤：In addition, the method for improving the sensitivity of PCR probe melting curve analysis of the present invention comprises the following steps:

A)引物探针、辅助序列设计；A) primer probe, auxiliary sequence design;

B)PCR扩增；B) PCR amplification;

C)熔解曲线分析。C) Melting curve analysis.

在步骤A的引物探针、辅助序列设计中，包括以下要点：In the primer probe and auxiliary sequence design of step A, the following points are included:

引物探针设计：采用本领域技术人员常用的引物探针软件设计引物探针；或者使用本发明的方法设计引物探针和不对称PCR体系。Primer probe design: use primer probe software commonly used by those skilled in the art to design primer probes; or use the method of the present invention to design primer probes and asymmetric PCR systems.

辅助序列的设计：Design of Auxiliary Sequences:

a.辅助序列为5-200个碱基的核酸序列，针对一个检测靶标可以包括多条辅助序列；a. The auxiliary sequence is a nucleic acid sequence of 5-200 bases, and a detection target can include multiple auxiliary sequences;

b.可与PCR产物的任何一条DNA链互补；b. Complementary to any DNA strand of the PCR product;

c.不能与PCR的引物、探针、其它辅助序列互补(例如连续6个以上碱基互补)；c. Cannot be complementary to primers, probes, and other auxiliary sequences of PCR (for example, complementary to more than 6 consecutive bases);

d.与引物、探针在与PCR产物杂交区域不重叠(每条辅助序列与引物或探针之间不能有例如连续6个以上碱基相同)；d. Do not overlap with primers and probes in the hybridization region with PCR products (for example, there can be no more than 6 consecutive bases identical between each auxiliary sequence and primers or probes);

e.优选的，为避免其被当做引物延伸，需对其3’端进行化学修饰，修饰方法包括但不限于3’磷酸化，3’采用双脱氧碱基，或用C3,或C6,C18修饰；e. Preferably, in order to prevent it from being used as primer extension, chemical modification of its 3' end is required. The modification methods include but are not limited to 3' phosphorylation, 3' using dideoxy base, or using C3, or C6, C18 modification;

f.优选的，辅助序列的Tm值高于探针的Tm值，还可以引入如LNA,MGB,PNA等碱基修饰以提高其Tm值。f. Preferably, the Tm value of the auxiliary sequence is higher than the Tm value of the probe, and base modifications such as LNA, MGB, PNA can also be introduced to increase its Tm value.

本发明进一步提供一种核酸检测试剂盒，该试剂盒包括：The present invention further provides a nucleic acid detection kit comprising:

在引物扩增区域内，针对非探针检测区域设计的辅助序列；In the primer amplification region, an auxiliary sequence designed for the non-probe detection region;

以及用于PCR扩增的DNA聚合酶和其它PCR组分如dNTP、镁离子。As well as DNA polymerase for PCR amplification and other PCR components such as dNTPs, magnesium ions.

本发明的有益效果：Beneficial effects of the present invention:

根据本发明，通过简单可行的体系设定，本发明解决了以下几个重要问题：According to the present invention, through simple and feasible system setting, the present invention solves the following important problems:

第一，解决了传统的Taqman技术不能在扩增后进行熔解曲线分析以进一步判断探针区域序列是否与目标序列完全一致的重要问题，让Taqman技术在PCR扩增后也能进行熔解曲线分析，赋予了Taqman技术新的生命力，实现了对Taqman技术的重要发展。First, it solves the important problem that the traditional Taqman technology cannot perform melting curve analysis after amplification to further determine whether the sequence of the probe region is completely consistent with the target sequence, so that Taqman technology can also perform melting curve analysis after PCR amplification, It gives new vitality to Taqman technology and realizes important development of Taqman technology.

第二，现有荧光PCR技术的一个重要缺陷是多重度低，一般一个通道检测一个靶标，常见的4通道荧光PCR仪只能检测4个靶标。采用多重探针熔解曲线法，理论上可以提高检测多重度。但是，多重探针熔解曲线分析技术开发难度大，体系不稳定，至今没有成熟和公开的技术，本发明在大量的研发中发现了一种可控制可重复并普遍适应的多重探针熔解曲线方法。Second, an important defect of the existing fluorescent PCR technology is the low multiplicity. Generally, one channel can detect one target, and the common 4-channel fluorescent PCR instrument can only detect four targets. Using the multiple probe melting curve method can theoretically improve the detection multiplex. However, the development of multiple probe melting curve analysis technology is difficult, the system is unstable, and there is no mature and disclosed technology so far. The present invention has found a controllable, repeatable and universally applicable multiple probe melting curve method in a lot of research and development. .

第三，对需要进行定量的目标序列，设计相应探针的Tm高于PCR的复性温度(三步法)，或复性与延伸温度(二步法)，并在此步骤采集荧光，PCR扩增体系采用无5’->3’外切酶活力的DNA聚合酶，发明人惊奇地发现这样也能达到实时监测PCR扩增过程的效果，(少数情况下，探针的熔解温度略低于PCR的退火温度时也能监测PCR的扩增过程)。这时的荧光的变化不是自淬灭探针的水解造成的，而是探针与目标序列杂交后荧光的增强所造成，这种荧光变化，虽然比Taqman技术中观察到的荧光变化要小很多，但我们大量的实验证明这种变化足以监测PCR扩增过程；且由于探针不被降解，探针浓度可以高于、等于、或低于高温引物，均可以获得扩增曲线和熔解曲线。更重要的是，我们发现从扩增曲线获得的Ct值，也与检测目标序列的浓度相关，能用于定量，如同Taqman技术一样。因此本发明形成了一种高多重定量检测技术，这种方法不使用具有5’->3’外切酶活力的DNA聚合酶，不属于Taqman技术，是对Taqman技术的超越。Third, for the target sequence that needs to be quantified, the Tm of the corresponding probe is designed to be higher than the renaturation temperature of PCR (three-step method), or the temperature of renaturation and extension (two-step method), and the fluorescence is collected in this step, PCR The amplification system uses a DNA polymerase without 5'->3' exonuclease activity. The inventor surprisingly found that this can also achieve the effect of real-time monitoring of the PCR amplification process (in a few cases, the melting temperature of the probe is slightly lower) The PCR amplification process can also be monitored at the annealing temperature of the PCR). The change in fluorescence at this time is not caused by the hydrolysis of the self-quenching probe, but is caused by the enhancement of fluorescence after the probe hybridizes to the target sequence. This fluorescence change, although much smaller than the fluorescence change observed in the Taqman technique , but our large number of experiments have proved that this change is sufficient to monitor the PCR amplification process; and since the probe is not degraded, the probe concentration can be higher, equal, or lower than the high temperature primer, and both amplification curves and melting curves can be obtained. More importantly, we found that the Ct value obtained from the amplification curve, which also correlates with the concentration of the detected target sequence, can be used for quantification, as with the Taqman technique. Therefore, the present invention forms a high multiplex quantitative detection technology, which does not use DNA polymerase with 5'->3' exonuclease activity, does not belong to Taqman technology, and surpasses Taqman technology.

第四，发明人发现通过混合使用具有5’->3’外切酶活力和不具有5’->3’外切酶活力的DNA聚合酶，并调节到合适的比例，扩增曲线的荧光能够进一步增强，同时熔解曲线并没有受到影响。形成一种定量和熔解曲线分析定性检测完美结合的方案。Fourth, the inventors found that by mixing the DNA polymerases with 5'->3' exonuclease activity and those without 5'->3' exonuclease activity, and adjusting to an appropriate ratio, the fluorescence of the amplification curve can be further enhanced, while the melting curve is not affected. It forms a perfect combination of quantitative and qualitative detection of melting curve analysis.

本发明可以用于对一个或多个目标序列同时进行定量检测、熔解曲线分析和基因分型。本发明特别适用于多重核酸检测，但也可以用于对一个目标序列同时进行定量检测和基因分型。The present invention can be used for simultaneous quantitative detection, melting curve analysis and genotyping of one or more target sequences. The present invention is particularly suitable for multiplex nucleic acid detection, but can also be used for simultaneous quantitative detection and genotyping of a target sequence.

第五，利用辅助序列，突破了传统的探针熔解曲线分析思维，进一步提高了探针熔解曲线分析的灵敏度。Fifth, the use of auxiliary sequences breaks through the traditional thinking of probe melting curve analysis and further improves the sensitivity of probe melting curve analysis.

附图说明Description of drawings

图1为高温引物、低温引物、探针工作原理图。Figure 1 is a schematic diagram of the working principle of high temperature primers, low temperature primers and probes.

图2为实施例1扩增曲线和熔解曲线。Figure 2 is the amplification curve and melting curve of Example 1.

图3为实施例2扩增曲线和熔解曲线。Figure 3 is the amplification curve and melting curve of Example 2.

图4为实施例3扩增曲线和熔解曲线。Figure 4 is the amplification curve and melting curve of Example 3.

图5为实施例4扩增曲线和熔解曲线。Figure 5 is the amplification curve and melting curve of Example 4.

实施例4的扩增曲线，其中有Ct值的，具有明显的S型扩增曲线；没有Ct值的，在图中表现为X轴上的平线，但有Tm值，可以定性不能定量。The amplification curve of Example 4, which has a Ct value, has an obvious S-shaped amplification curve; without a Ct value, it is shown as a flat line on the X-axis in the figure, but there is a Tm value, which can be qualitative but not quantitative.

实施例4的熔解曲线，各通道中，各型别HPV具有明显可区分的熔解曲线峰值，同一荧光通道内Tm值相差2℃以上，不会混淆。In the melting curve of Example 4, in each channel, each type of HPV has clearly distinguishable melting curve peaks, and the Tm values in the same fluorescence channel differ by more than 2°C, which will not be confused.

图6为辅助序列基本工作原理图。Figure 6 is a schematic diagram of the basic working principle of the auxiliary sequence.

图7为添加辅助序列前、实施例5、实施例6的扩增曲线和熔解曲线。FIG. 7 shows the amplification curves and melting curves of Example 5 and Example 6 before adding the helper sequence.

具体实施方式Detailed ways

以下通过实施例来进一步说明本发明。The present invention is further illustrated by the following examples.

实施例1Example 1

实验目的：利用本发明实现在Taqman技术检测后，进行熔解曲线分析的示例Experimental purpose: The present invention is used to realize an example of melting curve analysis after Taqman technology detection

经典的Taqman方法在扩增后不能进行熔解曲线分析，无法判断扩增片段是否与目标序列完全一致；利用本发明的设计方法(将PCR正向引物和反向引物设计成Tm值相差几度，在PCR扩增体系中低Tm值的引物(简称低温引物)浓度高于高Tm值的引物(简称高温引物)浓度，而且探针和高温引物与目标序列PCR产物的同一条链杂交(即探针设计在高温引物的目标链上))设计引物探针和浓度，可以在Taqman扩增后获得熔解曲线，如果多个反应中都可以得到正确、稳定的探针Tm值，则可以判断扩增片段在探针检测区域与目标序列完全一致。如果得到的Tm值小于探针Tm值，说明探针检测区域的序列与目标序列不完全一致。The classical Taqman method cannot carry out melting curve analysis after amplification, and cannot judge whether the amplified fragment is completely consistent with the target sequence; using the design method of the present invention (the PCR forward primer and reverse primer are designed so that the Tm value differs by a few degrees, In the PCR amplification system, the concentration of primers with low Tm values (referred to as low temperature primers) is higher than that of primers with high Tm values (referred to as high temperature primers), and the probe and high temperature primers hybridize to the same strand of the PCR product of the target sequence (ie, probe The probe is designed on the target strand of the high-temperature primer)) The primer probe and concentration can be designed, and the melting curve can be obtained after Taqman amplification. If correct and stable probe Tm values can be obtained in multiple reactions, the amplification can be judged. The fragment is completely identical to the target sequence in the detection region of the probe. If the obtained Tm value is less than the probe Tm value, it means that the sequence of the probe detection region is not completely consistent with the target sequence.

探针Tm值：扩增片段在探针检测区域与目标序列完全一致时的Tm；Probe Tm value: the Tm of the amplified fragment when the probe detection region is completely consistent with the target sequence;

Tm值稳定性：通过标准差SD体现，SD越小，稳定性越好。Tm value stability: reflected by the standard deviation SD, the smaller the SD, the better the stability.

实验设计：experimental design:

针对HPV33设计引物和Taqman探针，标记ROX荧光基团。序列在序列表中的编号分别是4、5、6。其中，引物4是高温引物，引物5是低温引物，探针6和高温引物目标链杂交，探针6的Tm值高于退火温度(60℃)，浓度高于引物4。采用有5’->3’外切酶活力的DNA聚合酶进行PCR扩增(本示例是实施例4的一个单项)。Primers and Taqman probes were designed for HPV33 and labeled with ROX fluorophore. The numbers of the sequences in the sequence listing are 4, 5, and 6, respectively. Among them, primer 4 is a high temperature primer, primer 5 is a low temperature primer, probe 6 hybridizes with the target strand of the high temperature primer, the Tm value of probe 6 is higher than the annealing temperature (60°C), and the concentration is higher than that of primer 4. PCR amplification was performed using a DNA polymerase with 5'->3' exonuclease activity (this example is a single item of Example 4).

实验条件：各组分及其在PCR反应体系中终浓度如下：(同实施例4，HPV33)Experimental conditions: each component and its final concentration in the PCR reaction system are as follows: (same as Example 4, HPV33)

注：引物Tm为设计温度，探针Tm为多次实际检测的正确的探针Tm值的平均值。Note: Primer Tm is the design temperature, and probe Tm is the average of the correct probe Tm values of multiple actual detections.

有5’->3’外切酶活性的DNA聚合酶来自市售TAKARA R001；PCR预混液来自楚天生物，货号Ctb104，含有镁离子，dNTP，buffer等PCR基本组分。The DNA polymerase with 5'->3' exonuclease activity is from the commercially available TAKARA R001; the PCR master mix is from Chutian Biology, the product number is Ctb104, which contains magnesium ions, dNTPs, buffer and other basic PCR components.

步骤B、C在PCR仪上的反应程序设置如下：(所有实施例均采用此程序设置)The reaction program settings of steps B and C on the PCR machine are as follows: (all embodiments adopt this program setting)

采用经测序验证HPV33为阳性的临床样本1例(3个浓度梯度)，实验数据如下：One case (3 concentration gradients) of clinical samples confirmed to be HPV33 positive by sequencing was used. The experimental data are as follows:

扩增曲线和熔解曲线原始图见附图2。The original graphs of amplification curves and melting curves are shown in Figure 2.

实验结果显示：实验结果具有良好的扩增曲线和正确、稳定的探针Tm值，可仅根据Tm值定性判断经扩增的HPV33片段与目标序列完全一致，进行HPV33定性检测。The experimental results show that the experimental results have a good amplification curve and a correct and stable probe Tm value, and the amplified HPV33 fragment can be qualitatively judged to be completely consistent with the target sequence only according to the Tm value, and the HPV33 qualitative detection can be carried out.

结论：采用本发明的方法(正向和反向引物的Tm值不同，引物在PCR扩增体系中低温引物浓度高于高温引物；探针和高温引物与目标序列的同一条链杂交；探针的浓度不低于高温引物的浓度，使用有5’->3’外切酶活力的DNA聚合酶)能够在Taqman探针法的基础上进行熔解曲线分析，即不仅获得了良好的扩增曲线，还获得了稳定的熔解曲线和正确的探针Tm值。本发明的方法解决了传统的Taqman技术不能在扩增后进行熔解曲线分析以进一步判断探针区域序列是否与目标序列完全一致的重要问题，是对Taqman技术的重要发展。Conclusion: Using the method of the present invention (the Tm values of the forward and reverse primers are different, the primer concentration in the PCR amplification system at low temperature is higher than that of the high temperature primer; the probe and the high temperature primer are hybridized with the same strand of the target sequence; the probe The concentration of the primer is not lower than that of the high-temperature primer, and the use of DNA polymerase with 5'->3' exonuclease activity) can perform melting curve analysis based on the Taqman probe method, that is, not only a good amplification curve is obtained. , a stable melting curve and correct probe Tm values were also obtained. The method of the invention solves the important problem that the traditional Taqman technology cannot perform melting curve analysis after amplification to further judge whether the sequence of the probe region is completely consistent with the target sequence, and is an important development of the Taqman technology.

实施例2Example 2

实验目的：使用无外切酶活性DNA聚合酶，不水解Taqman探针，不属于Taqman方法，仍然可以观察到扩增曲线和定量，并能进行熔解曲线分析。Experiment purpose: Using DNA polymerase without exonuclease activity, which does not hydrolyze Taqman probes, does not belong to the Taqman method, the amplification curve and quantification can still be observed, and melting curve analysis can be performed.

使用无外切酶活性DNA聚合酶，我们惊奇地发现这样也能达到实时监测PCR扩增过程的效果。少数情况下，探针的熔解温度略低于PCR的退火温度时也能监测PCR的扩增过程。这时的荧光的变化不是自淬灭探针的水解造成的，而是探针与目标序列杂交后荧光的增强所造成。这种荧光变化，虽然比Taqman技术中观察到的荧光变化要小很多，但我们大量的实验证明这种变化足以监测PCR扩增过程；且由于探针不被降解，探针浓度可以高于、等于、或低于高温引物，均可以获得扩增曲线和熔解曲线。更重要的是，我们发现从扩增曲线获得的Ct值，也与检测目标序列的浓度相关，能用于定量。因此本发明还形成了一种多重检测技术，它超越了Taqman技术。Using exonuclease-free DNA polymerases, we surprisingly found that it is also possible to monitor the PCR amplification process in real time. In rare cases, PCR amplification can also be monitored when the melting temperature of the probe is slightly lower than the annealing temperature of the PCR. The change in fluorescence at this time is not caused by the hydrolysis of the self-quenching probe, but by the enhancement of fluorescence after the probe hybridizes to the target sequence. Although this fluorescence change is much smaller than that observed in the Taqman technique, our extensive experiments have proved that this change is sufficient to monitor the PCR amplification process; and since the probe is not degraded, the probe concentration can be higher than, Amplification curves and melting curves can be obtained at or below the high temperature primers. More importantly, we found that the Ct value obtained from the amplification curve, which also correlates with the concentration of the detected target sequence, can be used for quantification. Thus the present invention also forms a multiplex detection technique that surpasses the Taqman technique.

实验设计：experimental design:

针对HPV16、33设计多重PCR引物和Taqman探针，分别标记VIC、ROX荧光基团。序列在序列表中的编号分别为1-6，其中，引物1为HPV16的高温引物，引物2为HPV16的低温引物，序列3为HPV16的探针；引物4为HPV33的高温引物，引物5为HPV33的低温引物，序列6为HPV33的探针；探针3、6均和高温引物目标链杂交，浓度不低于高温引物，探针Tm值均高于退火温度(60℃)。采用无5’->3’外切酶活力的DNA聚合酶进行PCR扩增。Multiplex PCR primers and Taqman probes were designed for HPV16 and 33, which were labeled with VIC and ROX fluorophores, respectively. The numbers of the sequences in the sequence table are 1-6 respectively, wherein, primer 1 is a high-temperature primer for HPV16, primer 2 is a low-temperature primer for HPV16, and sequence 3 is a probe for HPV16; primer 4 is a high-temperature primer for HPV33, and primer 5 is The low temperature primer of HPV33, sequence 6 is the probe of HPV33; probes 3 and 6 are hybridized with the target strand of the high temperature primer, the concentration is not lower than that of the high temperature primer, and the Tm value of the probe is higher than the annealing temperature (60°C). PCR amplification was performed using a DNA polymerase without 5'->3' exonuclease activity.

实验条件：各组分及其在多重PCR反应体系中终浓度如下：Experimental conditions: The components and their final concentrations in the multiplex PCR reaction system are as follows:

无5’->3’外切酶活性的DNA聚合酶来自楚天生物，货号Ctb113；PCR预混液来自楚天生物，货号Ctb104。The DNA polymerase without 5'->3' exonuclease activity is from Chutian Biotechnology, product number Ctb113; the PCR master mix is from Chutian Biotechnology, product number Ctb104.

步骤B、C在PCR仪上的反应程序设置同实施例1。The reaction program settings of steps B and C on the PCR machine are the same as those in Example 1.

采用HPV16、33国家标准品(中国食品药品检定研究院，注册检验用体外诊断试剂国家标准品，编号：360003)，稀释5个梯度，每个孔位分别加入一种HPV型别、一种浓度的样品，检测结果如下：HPV16 and 33 national standards (China National Institute for Food and Drug Control, National Standard for In Vitro Diagnostic Reagents for Registration Inspection, No.: 360003) were used, diluted in 5 gradients, and one HPV type and one concentration were added to each well. samples, the test results are as follows:

和熔解曲线原始图见附图3。

See Figure 3 for the original graph of the melting curve.

实验结果显示：HPV16、33进行5个梯度的浓度稀释，同一型别HPV不论浓度大小，具有正确、稳定的探针Tm值；根据Ct值得出线性关系方程，R ²大于0.977，线性关系良好。 The experimental results showed that HPV16 and 33 were diluted with 5 gradients of concentration, and the same type of HPV had correct and stable probe Tm value regardless of the concentration. According to the Ct value, the linear relationship equation was obtained, R ² was greater than 0.977, and the linear relationship was good.

结论：采用本发明的方法(正向和反向引物的Tm值不同，引物在PCR扩增体系中低温引物浓度高于高温引物；探针和高温引物与目标序列的同一条链杂交；探针的浓度不低于高温引物的浓度，使用无外切酶活性DNA聚合酶)，不采用有5’->3’外切酶活性DNA聚合酶，不水解Taqman探针，不属于Taqman方法，仍然可以观察到扩增曲线和熔解曲线，且适用于多种指标、多重核酸检测和定量。Conclusion: Using the method of the present invention (the Tm values of the forward and reverse primers are different, the primer concentration in the PCR amplification system at low temperature is higher than that of the high temperature primer; the probe and the high temperature primer are hybridized with the same strand of the target sequence; the probe The concentration is not lower than the concentration of high-temperature primers, using DNA polymerase without exonuclease activity), not using DNA polymerase with 5'->3' exonuclease activity, not hydrolyzing Taqman probes, not belonging to the Taqman method, still Amplification curves and melting curves can be observed and are suitable for multiple indicators, multiplex nucleic acid detection and quantification.

实施例3Example 3

实验目的：使用混合酶，可以克服使用无5’->3’外切酶活力的DNA聚合酶时，扩增熔解曲线信号很低，影响定量的问题。Experimental purpose: The use of mixed enzymes can overcome the problem of low amplification melting curve signal when using DNA polymerase without 5'->3' exonuclease activity, which affects quantification.

发明人在实践中发现，使用无5’->3’外切酶活力的DNA聚合酶这种方法在针对一些指标(如HPV52)时，扩增熔解曲线信号很低，影响定量。通过混合使用具有5’->3’外切酶活力和不具有5’->3’外切酶活力的DNA聚合酶，并调节到合适的比例，我们发现扩增曲线的荧光得到了增强，Ct值也有不同程度的变小，同时熔解曲线峰高未受到显著影响，不影响定性分析。The inventors found in practice that, when using a DNA polymerase without 5'->3' exonuclease activity, when targeting some indicators (such as HPV52), the amplification melting curve signal is very low, which affects the quantification. By mixing the DNA polymerases with 5'->3' exonuclease activity and those without 5'->3' exonuclease activity and adjusting to the appropriate ratio, we found that the fluorescence of the amplification curve was enhanced, The Ct value also decreased to varying degrees, and the peak height of the melting curve was not significantly affected, which did not affect the qualitative analysis.

实验设计：experimental design:

针对HPV52设计引物和Taqman探针，标记VIC荧光基团。序列在序列表中的编号分别是7、8、9。其中，引物8是低温引物，引物7是高温引物，探针和高温引物目标链杂交，探针9的Tm值低于退火温度(60℃)，浓度高于高温引物7。采用无5’->3’外切酶活力的DNA聚合酶(同实施例2)和有5’->3’外切酶活力的DNA聚合酶(同实施例1)按不同比例(无：有＝2:1；5:1；1)混合，进行PCR扩增。Primers and Taqman probes were designed for HPV52 and labeled with VIC fluorophore. The numbers of the sequences in the sequence listing are 7, 8, and 9, respectively. Among them, primer 8 is a low temperature primer, primer 7 is a high temperature primer, and the probe hybridizes with the target strand of the high temperature primer. Use DNA polymerase without 5'->3' exonuclease activity (same as Example 2) and DNA polymerase with 5'->3' exonuclease activity (same as Example 1) in different proportions (without: Yes = 2:1; 5:1; 1) mixed, and PCR amplification was performed.

实验条件：各组分及其在PCR反应体系中终浓度如下：Experimental conditions: The components and their final concentrations in the PCR reaction system are as follows:

采用经测序验证HPV52为阳性的临床样本检测，实验数据如下：The clinical samples confirmed by sequencing to be positive for HPV52 were tested. The experimental data are as follows:

扩增曲线和熔解曲线原始图见附图4。The original graphs of amplification curves and melting curves are shown in Figure 4.

实验结果显示：随着有5’->3’外切酶活力的DNA聚合酶的比例增加，Ct值逐渐减小，扩增曲线信号整体升高；Tm值稳定，均为正确的探针Tm值，但熔解曲线信号有所降低。当调节到合适比例(5:1)时，扩增曲线的荧光得到了增强，同时熔解曲线并没有受到太大影响，不影响定性。The experimental results show that: as the proportion of DNA polymerase with 5'->3' exonuclease activity increases, the Ct value gradually decreases, and the signal of the amplification curve increases as a whole; the Tm value is stable, all of which are correct probe Tm value, but the melting curve signal is reduced. When adjusted to an appropriate ratio (5:1), the fluorescence of the amplification curve was enhanced, and the melting curve was not greatly affected, which did not affect the characterization.

结论：使用本发明的方法(正向和反向引物的Tm值不同，引物在PCR扩增体系中低温引物浓度高于高温引物；探针和高温引物与目标序列的同一条链杂交；探针的浓度不低于高温引物的浓度，使用混合酶)，通过混合使用具有5’->3’外切酶活力和不具有5’->3’外切酶活力的DNA聚合酶，并调节到合适的比例，扩增曲线的荧光得到了增强，Ct值也有不同程度的变小，同时熔解曲线并没有受到显著影响。这是一种定量和熔解曲线分析定性检测完美结合的方案。Conclusion: Using the method of the present invention (the Tm values of the forward and reverse primers are different, the primer concentration of the low temperature primer is higher than that of the high temperature primer in the PCR amplification system; the probe and the high temperature primer are hybridized with the same strand of the target sequence; the probe The concentration is not lower than the concentration of high temperature primers, using mixed enzymes), by mixing the DNA polymerases with 5'->3' exonuclease activity and those without 5'->3' exonuclease activity, and adjusting to With a suitable ratio, the fluorescence of the amplification curve was enhanced, and the Ct value was also reduced to varying degrees, while the melting curve was not significantly affected. This is a perfect combination of quantitative and qualitative detection by melting curve analysis.

实施例4Example 4

实验目的：使用本发明的方法，开发简单通用的超多重PCR体系(本实施例是对实施例 1的补充)Experimental purpose: use the method of the present invention to develop a simple and general super multiplex PCR system (this example is a supplement to Example 1)

实验设计：experimental design:

物种：人、HPV病毒；指标：共9个，设计在一个PCR反应内，探针设计在高温引物目标链上；探针Tm值覆盖范围：40-80℃；探针共标记4种荧光，涉及4个荧光通道，每个通道2-3种指标，4个通道共检测9种检测序列。同一指标的高温引物和低温引物Tm值相差0.1-15℃，低温引物浓度：高温引物浓度约为1.05-30；同一通道探针Tm值两两之间相差2-35℃不等；优选，高温引物和低温引物Tm值相差3-10℃，探针Tm值覆盖范围：40-66.5℃；同一通道探针Tm值两两之间相差4-16℃；低温引物浓度：高温引物浓度约为1.3-25。优选探针浓度高于高温引物；探针Tm值有的高于、有的低于PCR退火步骤的温度，优选的，对于需要定量的指标，不低于PCR退火步骤的温度。Species: human, HPV virus; indicators: a total of 9, designed in one PCR reaction, the probe is designed on the target strand of the high temperature primer; the probe Tm value coverage: 40-80 ℃; the probe is labeled with 4 kinds of fluorescence, It involves 4 fluorescence channels, each channel has 2-3 indicators, and 4 channels detect a total of 9 detection sequences. The Tm values of high-temperature primers and low-temperature primers of the same index differ by 0.1-15℃, and the concentration of low-temperature primers: the concentration of high-temperature primers is about 1.05-30; the difference between the Tm values of probes in the same channel is 2-35℃; The Tm values of primers and low-temperature primers differ by 3-10°C, and the coverage range of probe Tm values is 40-66.5°C; the difference between the Tm values of probes in the same channel is 4-16°C; the concentration of low-temperature primers: the concentration of high-temperature primers is about 1.3 -25. Preferably, the concentration of the probe is higher than that of the high-temperature primer; the Tm value of the probe is higher or lower than the temperature of the PCR annealing step.

不同Tm引物和探针的设计表Design Tables for Different Tm Primers and Probes

注：引物Tm为设计温度，探针Tm为多次实际观测的温度平均值。Note: Primer Tm is the design temperature, and probe Tm is the average temperature of multiple actual observations.

其中，HPV16、33、66，B2M的探针Tm高于退火温度，HPV52、53、59、68、73的探针Tm低于退火温度。Among them, the probe Tm of HPV16, 33, 66, B2M was higher than the annealing temperature, and the probe Tm of HPV52, 53, 59, 68, 73 was lower than the annealing temperature.

步骤B、C在PCR仪上的反应程序同实施例1。The reaction procedures of steps B and C on the PCR machine are the same as those in Example 1.

采用经测序验证的对应指标为阳性的临床样本(1个样本3个浓度梯度，从上而下分别为组成浓度、稀释10倍、稀释100倍)，检测结果如下：Using clinical samples with positive corresponding indicators verified by sequencing (1 sample with 3 concentration gradients, from top to bottom, they are the composition concentration, 10-fold dilution, and 100-fold dilution), and the test results are as follows:

注：部分指标没有Ct值，该栏空白。Note: Some indicators have no Ct value, and the column is blank.

扩增曲线和熔解曲线原始结果见附图5。The original results of amplification curves and melting curves are shown in Figure 5.

检测结果显示：The test results show:

1.Tm值低于退火温度(60℃)的指标没有扩增曲线、没有Ct值，但有正确、稳定的探针Tm值，可仅根据探针Tm值判断经扩增的目标片段探针检测区域与目标序列完全一致，进行各指标定性检测。1. The index whose Tm value is lower than the annealing temperature (60°C) has no amplification curve and no Ct value, but has a correct and stable probe Tm value. The amplified target fragment probe can be judged only based on the probe Tm value. The detection area is completely consistent with the target sequence, and qualitative detection of each index is carried out.

2.Tm值高于退火温度的指标(60℃)的指标有较为理想的扩增曲线、Ct值，熔解曲线和正确、稳定的Tm值，可根据Tm值判断经扩增的目标片段探针检测区域与目标序列完全一致，进行各指标定性检测；以Ct值为基础，可以进行定量分析。2. The index whose Tm value is higher than the annealing temperature (60°C) has a relatively ideal amplification curve, Ct value, melting curve and correct and stable Tm value. The amplified target fragment probe can be judged according to the Tm value. The detection area is completely consistent with the target sequence, and qualitative detection of each index is carried out; based on the Ct value, quantitative analysis can be carried out.

结论：采用本发明的方法(正向和反向引物的Tm值不同，引物在PCR扩增体系中低温引物浓度高于高温引物；探针和高温引物与目标序列的同一条链杂交；探针的浓度不低于高温引物的浓度，使用有5’->3’外切酶活力的DNA聚合酶)能够在Taqman探针法的基础上进行熔解曲线分析，广泛适用于多种指标、多重核酸检测；同一指标具有正确、稳定的探针Tm值，可仅根据Tm值判断经扩增的目标片段探针检测区域与目标序列一致，进行各指标定性检测。对于探针Tm值高于PCR退火温度的指标，还具有良好的扩增曲线，可以进行定量分析。本发明的方法解决了高多重PCR中，传统的Taqman技术不能在扩增后进行熔解曲线分析以进一步判断探针区域序列是否与目标序列完全一致的重要问题。Conclusion: Using the method of the present invention (the Tm values of the forward and reverse primers are different, the primer concentration in the PCR amplification system at low temperature is higher than that of the high temperature primer; the probe and the high temperature primer are hybridized with the same strand of the target sequence; the probe The concentration of the primer is not lower than that of the high-temperature primer, and the use of DNA polymerase with 5'->3' exonuclease activity) can perform melting curve analysis on the basis of Taqman probe method, which is widely used in a variety of indicators, multiple nucleic acids Detection; the same index has a correct and stable probe Tm value, and the probe detection region of the amplified target fragment can be judged to be consistent with the target sequence only according to the Tm value, and qualitative detection of each index can be performed. For the index whose Tm value of the probe is higher than the PCR annealing temperature, it also has a good amplification curve and can be quantitatively analyzed. The method of the invention solves the important problem that in high multiplex PCR, the traditional Taqman technology cannot perform melting curve analysis after amplification to further judge whether the probe region sequence is completely consistent with the target sequence.

实施例5Example 5

实验目的：在本发明独特的引物探针设计基础上，采用辅助序列，可提高探针熔解曲线分析的灵敏度。Experimental purpose: On the basis of the unique primer probe design of the present invention, the use of auxiliary sequences can improve the sensitivity of probe melting curve analysis.

发明人在PCR-熔解曲线产品开发实践过程中，发现人类G6PD基因SNP位点rs398123546经过多次设计开发均无法获得理想的实验结果，具体表现为：用测序确定的野生型、杂合子和突变型，用本实验无法区分。由于有一个正确、稳定的熔解曲线峰，排除了引物、探针和PCR体系的问题，判断是突变型的检出灵敏度问题。During the practice of PCR-melting curve product development, the inventor found that the SNP site rs398123546 of the human G6PD gene could not obtain ideal experimental results after many times of design and development. , cannot be distinguished by this experiment. Since there is a correct and stable melting curve peak, the problems of primers, probes and PCR system are ruled out, and it is judged that it is the detection sensitivity of mutant type.

利用本发明步骤A的方法，设计引物、探针、辅助序列，在PCR体系中的终浓度如下：Using the method of step A of the present invention, primers, probes and auxiliary sequences are designed, and the final concentrations in the PCR system are as follows:

步骤B、C在PCR仪上的反应程序设置如下：The reaction program settings of steps B and C on the PCR machine are as follows:

检测结果如下：The test results are as follows:

扩增曲线和熔解曲线见附图7。The amplification curve and melting curve are shown in Figure 7.

实验结果表明：加辅助序列后测得两个稳定的熔解曲线峰，突变型Tm为50.5℃左右，野生型为57℃左右。The experimental results showed that two stable melting curve peaks were measured after adding the auxiliary sequence, the Tm of the mutant type was about 50.5℃, and that of the wild type was about 57℃.

未加辅助序列时，野生型能够稳定检出，但突变型无法检出，加辅助序列后，实施例5的野生型和突变型Tm都能稳定检出，表明辅助序列有效地克服了阻碍突变等位基因检出的因素，增强了突变等位基因的检测灵敏度。Without the helper sequence, the wild type can be stably detected, but the mutant type cannot be detected. After the helper sequence is added, both the wild type and mutant Tm of Example 5 can be stably detected, indicating that the helper sequence effectively overcomes the impeding mutation. Allele detection factors enhance the detection sensitivity of mutant alleles.

实施例6Example 6

实验目的：采用辅助序列，在对称PCR中也可实现提高探针熔解曲线分析的灵敏度。Experimental purpose: The use of auxiliary sequences can also improve the sensitivity of probe melting curve analysis in symmetric PCR.

发明人在实验过程中发现，使用辅助序列，并不一定需要设计成本发明权利要求1所述的不对称PCR，对称PCR也可行。In the course of experiments, the inventors found that it is not necessary to design the asymmetric PCR described in claim 1 of the present invention by using the auxiliary sequence, and symmetric PCR is also feasible.

利用本发明一种提高PCR熔解曲线分析灵敏度的方法，设计引物、探针、辅助序列，检测对象、引物、探针、辅助序列的序列设计同实施例5，采用对称PCR设计，各组分在PCR体系中的终浓度如下：Using a method of the present invention to improve the sensitivity of PCR melting curve analysis, primers, probes, and auxiliary sequences are designed. The sequence design of detection objects, primers, probes, and auxiliary sequences is the same as that in Example 5. Symmetric PCR design is adopted. The final concentrations in the PCR system are as follows:

检测结果如下：The test results are as follows:

实验结果表明：加辅助序列后，实施例6的野生型和突变型Tm都能稳定检出，Tm值与实施例5一致，表明辅助序列有效地克服了阻碍突变等位基因检出的因素，增强了突变等位基因的检测灵敏度，辅助序列功能的实现不依赖于不对称PCR设计。The experimental results show that: after adding the auxiliary sequence, the wild-type and mutant Tm of Example 6 can be stably detected, and the Tm value is consistent with that of Example 5, indicating that the auxiliary sequence effectively overcomes the factors that hinder the detection of mutant alleles. The detection sensitivity of mutant alleles is enhanced, and the realization of helper sequence function does not depend on asymmetric PCR design.

上述实施例仅为说明本发明，不能以此限定本发明的保护范围。应当理解，本发明提供的引物浓度要求、探针浓度要求、上下游引物倍数差异要求、Tm值要求、各Tm值之间的差异要求、混合酶比例要求相当宽泛，能够满足绝大多数核酸检测的引物探针设计需求，绝非仅适合于实施例中的引物探针。The above-mentioned embodiments are only for illustrating the present invention, and cannot limit the protection scope of the present invention. It should be understood that the primer concentration requirements, probe concentration requirements, upstream and downstream primer fold difference requirements, Tm value requirements, difference requirements between Tm values, and mixed enzyme ratio requirements provided by the present invention are quite broad, and can meet the requirements of most nucleic acid detection. The design requirements of primers and probes are not only suitable for the primers and probes in the examples.

Claims

一种基于探针熔解曲线分析的多重核酸检测方法，所述方法包括以下步骤：A multiple nucleic acid detection method based on probe melting curve analysis, the method comprises the following steps:

步骤A：不对称PCR体系设计；Step A: Design of asymmetric PCR system;

步骤B：PCR扩增；Step B: PCR amplification;

步骤C：熔解曲线分析；Step C: melting curve analysis;

所述步骤A的不对称PCR体系设计包括：The asymmetric PCR system design of the step A includes:

A1.针对每个目标核酸序列设计正向和反向PCR引物，每个目标序列设计至少一对引物，针对多个目标核酸序列设计简并引物，或设计多条引物；A1. Design forward and reverse PCR primers for each target nucleic acid sequence, design at least one pair of primers for each target sequence, design degenerate primers for multiple target nucleic acid sequences, or design multiple primers;

A2.对每个目标核酸序列设计一条探针，探针设计位置位于正向和反向引物之间；A2. Design a probe for each target nucleic acid sequence, and the probe design position is located between the forward and reverse primers;

其中A1中正向和反向引物的Tm值不同，引物在PCR扩增体系中低温引物浓度高于高温引物；A2中探针和高温引物与目标序列的同一条链杂交。The Tm values of the forward and reverse primers in A1 are different, and the primers in the PCR amplification system have a higher concentration of low-temperature primers than high-temperature primers; in A2, the probe and high-temperature primers hybridize to the same strand of the target sequence.
根据权利要求1所述的多重核酸检测方法，其特征是，A1中，高温和低温引物的Tm值相差0.1-15℃，优选高温引物和低温引物Tm值相差3-10℃；低Tm引物浓度与高Tm引物浓度的比值为1.05至30，优选的，1.3至25；A2中，探针Tm范围在40-80℃；同一通道相邻的Tm值的差值在2-35℃，优选探针Tm值两两之间相差2.5-16℃。The multiplex nucleic acid detection method according to claim 1, wherein, in A1, the Tm values of the high temperature primers and the low temperature primers differ by 0.1-15°C, preferably the Tm values of the high temperature primers and the low temperature primers differ by 3-10°C; the low Tm primer concentration The ratio to the high Tm primer concentration is 1.05 to 30, preferably 1.3 to 25; in A2, the probe Tm range is 40-80 °C; the difference between adjacent Tm values in the same channel is 2-35 °C, preferably the probe The difference between the needle Tm values is 2.5-16°C.
根据权利要求1所述的多重核酸检测方法，其特征是，所述方法用于1-3个核苷酸突变、***或缺失的核酸检测，对每个目标突变位点的野生型和突变型仅设计一条探针；设计的探针在与野生型目标序列和突变的目标序列分别结合，野生型目标序列和突变的目标序列与探针分别结合的Tm值相差2-30℃；The multiplex nucleic acid detection method according to claim 1, wherein the method is used for nucleic acid detection of 1-3 nucleotide mutations, insertions or deletions. Only one probe is designed; the designed probe binds to the wild-type target sequence and the mutated target sequence, respectively, and the Tm values of the wild-type target sequence and the mutated target sequence and the probe are different by 2-30°C;

或or

所述的方法用于≥3个碱基差别的核酸检测，其中对野生型序列和突变序列分别设计探针，分别设计的探针Tm值相差2-30℃。The method is used for nucleic acid detection with a difference of ≥3 bases, wherein probes are respectively designed for the wild-type sequence and the mutant sequence, and the Tm values of the respectively designed probes differ by 2-30°C.
根据权利要求1所述的多重核酸检测方法，其特征是：探针标记有荧光发色基团和荧光淬灭基团，荧光发色基团和淬灭基团标记在探针的两端或非两端位置。The multiple nucleic acid detection method according to claim 1, wherein the probe is labeled with a fluorescent chromophore group and a fluorescent quencher group, and the fluorescent chromophore group and the quencher group are labeled on both ends of the probe or Not at both ends.
根据权利要求1所述的多重核酸检测方法，其特征是，步骤B中，用于PCR扩增的DNA聚合酶是无5’->3’外切酶活力的聚合酶，或有5’->3’外切酶活力的聚合酶，或两者的混合物；优选地，当PCR扩增采用具有5’->3’外切酶活力的聚合酶时，探针的浓度至少不能低于高温引物的浓度。The multiplex nucleic acid detection method according to claim 1, wherein in step B, the DNA polymerase used for PCR amplification is a polymerase without 5'->3' exonuclease activity, or a polymerase with 5'->3' exonuclease activity. A polymerase with >3' exonuclease activity, or a mixture of the two; preferably, when a polymerase with 5'->3' exonuclease activity is used for PCR amplification, the concentration of the probe should at least not be lower than the high temperature primer concentration.
根据权利要求5所述的多重核酸检测方法，其特征是，针对需要进行定量检测的目标序列的检测探针，其Tm值至少不能低于PCR退火步骤的温度。The multiplex nucleic acid detection method according to claim 5, wherein the detection probe for the target sequence that needs to be quantitatively detected has a Tm value at least not lower than the temperature of the PCR annealing step.
根据权利要求1所述的多重核酸检测方法，其特征是，在熔解曲线分析步骤C时，在PCR扩增步骤结束后在进行从低温到高温的熔解曲线分析前，包括一个缓慢的从高温到低温的 DNA从单链形成双链的复性步骤，降温速度低于8℃/秒，优选的低于3℃/秒，更优选的低于2℃/秒；或分阶段降温，94℃至80℃，常规快速降温，从80℃至45℃进行缓慢降温。The multiplex nucleic acid detection method according to claim 1, wherein in the melting curve analysis step C, after the PCR amplification step is completed, before the melting curve analysis from low temperature to high temperature is performed, a slow transition from high temperature to high temperature is included. In the renaturation step of DNA at low temperature from single strand to double strand, the cooling rate is lower than 8°C/sec, preferably lower than 3°C/sec, more preferably lower than 2°C/sec; or cooling in stages, 94°C to 80°C, conventional rapid cooling, and slow cooling from 80°C to 45°C.
根据权利要求1所述的多重核酸检测方法，其特征是，可用于对多个目标序列进行多重检测，或对一个目标序列进行单重检测。The multiplex nucleic acid detection method according to claim 1, characterized in that it can be used for multiplex detection of multiple target sequences, or single-plex detection of one target sequence.
根据权利要求1所述的方法，其特征是：使用辅助序列，所述辅助序列为长度5-200个碱基的序列，且辅助序列与检测探针在与PCR产物杂交区域不重叠，优选地，所述辅助序列3’端进行修饰以阻止其被延长。The method according to claim 1, wherein an auxiliary sequence is used, the auxiliary sequence is a sequence of 5-200 bases in length, and the auxiliary sequence and the detection probe do not overlap in the hybridization region with the PCR product, preferably , the 3' end of the helper sequence is modified to prevent it from being elongated.
一种基于探针熔解曲线分析的核酸检测试剂盒，该试剂盒包括：A nucleic acid detection kit based on probe melting curve analysis, the kit includes:

针对每个目标核酸序列设计的正向和反向PCR引物，每个目标序列设计至少一对引物，针对多个目标核酸序列可以设计简并引物，或设计多条引物；Forward and reverse PCR primers designed for each target nucleic acid sequence, at least one pair of primers designed for each target sequence, degenerate primers can be designed for multiple target nucleic acid sequences, or multiple primers can be designed;

针对每个目标核酸序列设计的一条探针，探针设计位置位于正向和反向引物之间，针对不同目标序列的探针Tm值不同；A probe designed for each target nucleic acid sequence, the probe design position is located between the forward and reverse primers, and the probe Tm values for different target sequences are different;

以及用于PCR扩增的dNTP和DNA聚合酶、镁离子等常规组分；And conventional components such as dNTP and DNA polymerase, magnesium ions for PCR amplification;

其中正向和反向引物的Tm值不同，引物在PCR扩增体系中Tm值低的引物浓度高于Tm值高的引物；探针和Tm值高的引物与目标序列的同一条链杂交；Among them, the Tm values of the forward and reverse primers are different, and the primer concentration of the primer with a low Tm value is higher than that of the primer with a high Tm value in the PCR amplification system; the probe and the primer with a high Tm value hybridize to the same strand of the target sequence;

优选地，高温和低温引物的Tm值相差0.1-15℃；低Tm引物浓度与高Tm引物浓度的比值为1.05至30；A2步骤中，探针Tm范围在40-80℃；相邻的Tm值的差值在2-35℃；Preferably, the Tm values of the high temperature and low temperature primers differ by 0.1-15°C; the ratio of the low Tm primer concentration to the high Tm primer concentration is 1.05 to 30; in step A2, the probe Tm range is 40-80°C; adjacent Tm The difference between the values is 2-35°C;

优选地，DNA聚合酶为无5’->3’外切酶活力的聚合酶，或有5’->3’外切酶活力的聚合酶，或两者的混合物。Preferably, the DNA polymerase is a polymerase without 5'->3' exonuclease activity, or a polymerase with 5'->3' exonuclease activity, or a mixture of the two.
一种提高PCR熔解曲线分析灵敏度的方法，包括以下步骤：A method for improving the sensitivity of PCR melting curve analysis, comprising the following steps:

步骤A：引物、探针、辅助序列设计；Step A: primer, probe, auxiliary sequence design;

步骤B：PCR扩增；Step B: PCR amplification;

步骤C：熔解曲线分析；Step C: melting curve analysis;

其特征在于，反应体系使用了辅助序列。It is characterized in that the reaction system uses an auxiliary sequence.
根据权利要求11所述的方法，其特征是：步骤A中所述辅助序列是一条或多条长度为5-200个碱基的核酸链，采用多条辅助序列时，可与PCR产物的任何一条DNA链互补；辅助序列不能与PCR的引物、探针、其它辅助序列互补；辅助序列与引物、探针在与PCR产物杂交区域不重叠；优选的，辅助序列的Tm值高于检测探针的Tm值，优选的，还可以引入如LNA,MGB,PNA等碱基修饰。The method according to claim 11, wherein the auxiliary sequence in step A is one or more nucleic acid chains with a length of 5-200 bases. A DNA strand is complementary; the auxiliary sequence cannot be complementary to the primers, probes, and other auxiliary sequences of PCR; the auxiliary sequence does not overlap with the primers and probes in the hybridization area with the PCR product; preferably, the Tm value of the auxiliary sequence is higher than that of the detection probe. Preferably, base modifications such as LNA, MGB, and PNA can also be introduced.
根据权利要求11所述的方法，其特征是，对所述辅助序列3’端进行修饰以阻止其被延长。The method of claim 11, wherein the 3' end of the helper sequence is modified to prevent it from being elongated.
一种核酸检测试剂盒，其特征是，使用了辅助序列，所述辅助序列是一条或多条长度为5-200个碱基的核酸链，采用多条辅助序列时，可与PCR产物的任何一条DNA链互补；辅助序列不能与PCR的引物、探针、其它辅助序列互补；辅助序列与引物、探针在与PCR产物杂交区域不重叠；优选的，辅助序列的Tm值高于检测探针的Tm值，优选的，还可以引入如LNA、MGB、PNA等碱基修饰。A nucleic acid detection kit is characterized in that an auxiliary sequence is used, and the auxiliary sequence is one or more nucleic acid chains with a length of 5-200 bases. A DNA strand is complementary; the auxiliary sequence cannot be complementary to the primers, probes, and other auxiliary sequences of PCR; the auxiliary sequence does not overlap with the primers and probes in the hybridization area with the PCR product; preferably, the Tm value of the auxiliary sequence is higher than that of the detection probe. Preferably, base modifications such as LNA, MGB, and PNA can also be introduced.