WO2022068079A1 - 一种基于微滴式数字pcr技术的乙型肝炎病毒t216c突变检测方法 - Google Patents

一种基于微滴式数字pcr技术的乙型肝炎病毒t216c突变检测方法 Download PDF

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WO2022068079A1
WO2022068079A1 PCT/CN2020/136277 CN2020136277W WO2022068079A1 WO 2022068079 A1 WO2022068079 A1 WO 2022068079A1 CN 2020136277 W CN2020136277 W CN 2020136277W WO 2022068079 A1 WO2022068079 A1 WO 2022068079A1
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hbv
digital pcr
mutation
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任艳丽
陈智
朱海红
洪萌
吴国娣
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Zhejiang University
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  • the invention belongs to the technical field of biological detection methods, in particular to a method for detecting the mutation of hepatitis B virus T216C based on droplet digital PCR technology.
  • Hepatitis B is still a worldwide public health problem, and hepatitis B virus (HBV)-related acute on chronic liver failure (ACLF) is an important cause of death in patients with chronic hepatitis .
  • HBV hepatitis B virus
  • ACLF chronic liver failure
  • HBV is a member of the hepadnavirus family, and its genome is about 3200 nucleotides in length and contains 4 overlapping open reading frames, encoding HBV surface antigen, core antigen, polymerase and multifunctional non- Structural protein X.
  • the mutation rate of HBV is significantly higher than that of other DNA viruses.
  • the mutation rate of the HBV genome can increase with the replication of the virus, and some of these mutations may be associated with the acute exacerbation of chronic hepatitis, which can be used as early warning biomarkers of ACLF and targets for antiviral therapy.
  • Some studies collected ACLF samples and screened HBV mutations related to the pathogenesis of ACLF by HBV whole-genome sequencing, and found that the T216C mutation was an independent risk factor for the occurrence of ACLF and could be used as a molecular marker for predicting the exacerbation of hepatitis B. Provide a basis for improving the prognosis of HBV-infected patients.
  • Digital PCR Digital PCR
  • Digital PCR is an absolute quantification technology of nucleic acid molecules. It does not rely on standard curves and reference samples to directly detect the copy number of target sequences. Compared with traditional fluorescence quantification, digital PCR has higher sensitivity and specificity. sex and precision.
  • digital PCR Since digital PCR only judges the presence or absence of two amplification states when interpreting the results, it does not need to detect the intersection of the fluorescent signal and the set threshold line, and does not depend on the Ct value at all, so the digital PCR reaction is greatly affected by the amplification efficiency Reduced, the tolerance to reaction inhibitors is greatly improved; the process of digital PCR standard reaction system allocation can greatly reduce the concentration of background sequences that compete with target sequences, so digital PCR technology is suitable for detection in complex backgrounds Rare mutations have obvious advantages in the detection of very small nucleic acid samples, the detection of rare mutations in complex backgrounds, and the identification of small differences in expression.
  • the droplet digital PCR instrument forms thousands of nano-liter droplets from the reaction system containing nucleic acid molecules, each of which either contains no nucleic acid target molecules to be detected, or contains one to several nucleic acid targets to be detected. molecules, and each droplet acts as an independent PCR reactor.
  • a droplet analyzer is used to detect each droplet.
  • the droplet with fluorescent signal is interpreted as 1, and the droplet without fluorescent signal is interpreted as 0.
  • the detection molecule can be calculated and given. concentration or copy number.
  • the amplification refractory mutation system was first established by Newton et al. to detect known mutations. Under certain conditions, the mismatch at the 3' end of PCR primers leads to a sharp decrease in the product. For different known mutations, designing appropriate primers can directly achieve the purpose of distinguishing mutant and wild-type genes by PCR method. ARMS technology is to design primers for the mutation site, and place the mutation site to be detected at the 3' end of the primer. When the mutation primer amplifies the wild-type template, it will be blocked and cannot be effectively amplified. This ARMS technology has high specificity and high sensitivity, and has been used for the detection of point mutations in various diseases.
  • the invention combines a mutation amplification block system and a digital PCR system to develop a mutation detection method with high sensitivity and high specificity that can be absolutely quantitative.
  • the purpose of the present invention is to provide a method for detecting the mutation site of HBV T216C based on the droplet digital PCR technology.
  • the present invention provides a method for detecting HBV T216C mutation based on droplet digital PCR technology, the method comprising the following steps:
  • the present invention provides primers and probes for detecting the HBV gene T216C mutation site in step (1), including the forward primer shown in SEQ NO: 1, the reverse primer shown in SEQ ID: 2, and the forward primer shown in SEQ ID: 2. Probe indicated by NO:3.
  • the present invention provides primers and probes for step (2) to detect the conserved sequence of HBV gene, including the forward primer shown by SEQ NO:4, the reverse primer shown by SEQ ID:5, and the reverse primer shown by SEQ ID NO:6 probe shown.
  • the detection probe of the present invention is labeled with a fluorescent dye at the 5' end, and the labeled fluorescent group is preferably any one of FAM, VIC or HEX, preferably FAM fluorescence, and the 3' end is labeled with a quenching group.
  • the group is any one of BHQ1, BHQ2 or MGB, preferably BHQ1.
  • the detection probe of the present invention is labeled with a fluorescent dye at the 5' end, and the labeled fluorescent group is preferably any one of FAM, VIC or HEX, preferably VIC fluorescence, and the 3' end is labeled with a quenching group.
  • the group is any one of BHQ1, BHQ2 or MGB, preferably BHQ1.
  • the digital PCR detection reagent for the two-channel detection in step (3) of the present invention further includes a droplet generation card, a 96-well reaction plate, an aluminum foil heat-sealing film and a reading oil.
  • the droplet digital PCR detection method of the present invention at least comprises the following steps:
  • microdroplet digital PCR amplification reaction conditions of the present invention are as follows: 95°C for 10 minutes, then 94°C for 30 seconds, 58°C for 45 seconds, a total of 40 cycles, and finally 98°C for 10 minutes for inactivation, Cool down to 4°C.
  • the present invention has the following advantages and effects:
  • the present invention combines the digital PCR technology and the mutation amplification block system method, and has the characteristics of high sensitivity of the digital PCR technology, absolute quantification and strong mutation specificity detection by the mutation amplification block system method. Specific detection when the abundance of site mutation genes is very low can accurately and quickly obtain the proportion of low-abundance mutations, which can early warning and predict the severity of hepatitis B, and provide a basis for preventing the onset of ACLF and improving the prognosis of HBV-infected patients. .
  • the present invention is based on the digital PCR system platform, and has the advantage of absolute quantification, and the mutation ratio can be directly calculated according to the result.
  • the detection method of the present invention is simple to operate, has high compatibility with traditional fluorescence quantitative PCR technology, and has good consistency of detection results, and has high practical application value.
  • Figure 1 A plot of detection of negative for mutant and positive for wild-type.
  • Figure 2 The detection diagram of the ratio of mutant plasmid template to wild plasmid template is 1:1000.
  • ARMS method For the mutation of HBV216 site, according to the principle of ARMS method, use Beacon Designer biological software to design primers and probes, use primer design rules, manually input the upstream primer containing the mutation site, and place the mutation site at the 3' end of the upstream primer, The software searches for matching downstream primers and probes; the forward primer shown in SEQ NO: 1, the reverse primer shown in SEQ ID: 2, the probe shown in SEQ ID NO: 3 are obtained, and the probe fluorophore
  • the group is FAM and the quenching group is BHQ1.
  • the primers and probes of the present invention are optimized from multiple primers and probes, and have the characteristics of strong specificity and high sensitivity.
  • test sample mix the plasmid containing the HBV mutant gene (T216C) and the plasmid containing the HBV conserved sequence according to the copy number content of 1:100, 1:1000 and 1:5000, and take 2ul after mixing. DNA testing.
  • ddPCR reaction solution 2*ddPCR supermix for probes(no dUTP)(BIO-RAD), used to detect the upstream and downstream primers and probes of the 216 mutation site, used to detect the upstream and downstream primers and probes of HBV conserved sequences Needle, mixed DNA template to be tested, distilled water was supplemented to a total volume of 20ul, and a ddPCR reaction solution was prepared, in which the concentrations of the two pairs of upstream and downstream primers were both 500nM, and the content of the two probes was 250nM.

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Abstract

一种基于微滴式数字PCR技术的乙型肝炎病毒T216C突变检测方法。结合微滴式数字PCR技术和突变扩增系统方法，利用数字PCR的高灵敏度和绝对定量的优势，联合突变扩增阻滞系统的高特异性优势，开发一个能够早期检测低丰度HBV突变情况，以便更好的满足临床用药及科研的需求。

Description

一种基于微滴式数字PCR技术的乙型肝炎病毒T216C突变检测方法 技术领域
本发明属于生物检测方法技术领域，具体涉及一种基于微滴式数字PCR技术的乙型肝炎病毒T216C突变检测方法。
背景技术
乙型病毒性肝炎仍是一个世界性的公共卫生问题，乙型肝炎病毒(Hepatitis B virus,HBV)相关的慢加急性肝衰竭(acute on chromc liver failure，ACLF)是慢性肝炎患者死亡的重要原因。长期以来宿主免疫一直被认为是乙型肝炎加剧的主要原因，但是近年的研究结果表明病毒因素在ACLF的发病中同样起重要作用。HBV是嗜肝病毒家族的一员，其基因组全长约为3200个核苷酸，包含4个相互重叠的开放性读码框，分别编码HBV表面抗原、核心抗原、聚合酶和多功能的非结构性蛋白X。由于HBV的复制过程包含前基因组RNA的逆转录，其逆转录酶缺乏校正功能，故HBV的突变率显著高于其它的DNA病毒。HBV基因组的突变率可随着病毒的复制不断增加，其中一些突变可能与慢性肝炎的急性加剧相关，可作为ACLF的预警的生物标志物和抗病毒治疗的靶标。有研究通过收集ACLF样本，利用HBV全基因组测序筛选出与ACLF发病相关的HBV突变，发现T216C突变为ACLF发生的独立危险因素，可作为预测乙型肝炎加剧的分子标志物，为阻止ACLF发病和改善HBV感染者的预后提供依据。
数字PCR(Digital PCR)技术是一种核酸分子绝对定量技术，不依赖于标准曲线和参照样本，直接检测目标序列的拷贝数，与传统的荧光定量相比，数字PCR有更高的灵敏度、特异性和精确性。由于数字PCR在进行结果判读时仅判断有/无两种扩增状态，不需要检测荧光信号与设定阈值线的交点，完全不依赖于Ct值，因此数字PCR反应受扩增效率的影响大大降低，对反应抑制物的耐受能力大大提高；数字PCR标准反应体系分配的过程可以极大程度上降低与目标序列有竞争性作用的背景序列浓度，因此数字PCR技术适用于在复杂背景中检测稀有突变，在极微量核酸样本检测、复杂背景下稀有突变检测和表达量微小差异鉴定方面具有明显的优势。微滴式数字PCR仪是将含有核酸分子的反应体系形成成千上万个纳升级的微滴，其中每个微滴或不含待检核酸靶分子，或者含有一个至数个待检核酸靶分子，且每个微滴都作为一个独立的PCR反应器。经PCR扩增后，采用微滴分析仪对每个微滴进行检测，有荧光信号的微滴判读为1，没有荧光信号的微滴判读为0，根据泊松分布原理可计算给出检测分子的浓度或拷贝数。
突变扩增阻滞系统(amplification refractory mutation system,ARMS)是Newton等首先建立用来检测已知突变的方法。在一定条件下，PCR引物3'未端的错配导致产物的急剧减少,针对不同的已知突变，设计适当的引物可以通过PCR方法直接达到区分突变型和野生型基因的目的。ARMS技术即针对突变位点设计引物，将需检测的突变位点置于引物的3’端，突变引物扩增野生型模板时会被阻滞，不能有效扩增。该系ARMS技术具有高特异性和高灵敏度，已用于多种疾病的点突变的检测。
本发明将突变扩增阻滞系统和数字PCR系统结合起来，开发具有高灵敏度和高特异性的能够绝对定量的突变检测方法。
发明内容
针对现有技术存在的问题和需求，本发明目的是提供一种基于微滴式数字PCR技术的HBV T216C突变位点检测方法。结合微滴式数字PCR技术和突变扩增系统方法，利用数字PCR的高灵敏度和绝对定量的优势，联合突变扩增阻滞系统的高特异性优势，开发一个能够早期检测低丰度HBV突变情况，以便更好的满足临床用药及科研的需求。
技术方案：为实现上述目的，本发明提供一种基于微滴式数字PCR技术检测HBV T216C突变的方法，所述方法包括如下步骤：
(1)对HBV基因的T216C突变位点设计PCR扩增引物和探针
(2)对HBV基因保守序列设计PCR扩增引物和探针
(3)对步骤(1)和(2)的突变位点基因和保守序列基因进行两通道的微滴式数字PCR检测
(4)通过对HBV突变位点的微滴式数字PCR扩增结果确定待测样品是否存在突变及突变拷贝数，通过对HBV基因保守序列基因的数字PCR扩增结果确定样品中HBV基因总数，通过比较来计算判断HBV突变基因占HBV基因总数的比例。
本发明提供了用于步骤(1)检测HBV基因T216C突变位点的引物和探针包括由SEQ NO:1所示的正向引物、由SEQ ID:2所示的反向引物、由SEQ ID NO:3所示的探针。
NO:1 HBV基因T216C突变位点正向引物
GTTACAGGCGGGGTTTTTCGC
NO:2 HBV基因T216C突变位点反向引物
TTGGGGACTGCGAATTTTGG
NO:3 HBV基因T216C突变位点探针
FAM-TCTAGACTCGTGGTGGACTTCTCTCA-BHQ1
本发明提供用于步骤(2)检测HBV基因保守序列的引物和探针包括由SEQ NO:4示的正向引物、由SEQ ID:5所示的反向引物、由SEQ ID NO:6所示的探针。
NO:4 HBV基因保守区域正向引物
CCTGGCTATCGCTGGATGTGT
NO:5 HBV基保守区域反向引物
GGACAAACGGGCAACATACCTT
NO:6 HBV基因保守区域探针
VIC-CCTCTTCATCCTGCTGCTATGCCTCA-BHQ1
本发明所述检测探针5’端标记荧光染料，标记荧光基团优选的是FAM、VIC或HEX中的任意一种，优选为FAM荧光，3’端标记淬灭基团，所述淬灭基团为BHQ1、BHQ2或MGB中的任意一种，优选为BHQ1。
本发明所述检测探针5’端标记荧光染料，标记荧光基团优选的是FAM、VIC或HEX中的任意一种，优选为VIC荧光，3’端标记淬灭基团，所述淬灭基团为BHQ1、BHQ2或MGB中的任意一种，优选为BHQ1。
本发明所述用于步骤(3)两通道检测的数字PCR检测试剂，还包括微滴生成卡，96孔反应板、铝箔热封膜和读数油。
本发明所述微滴式数字PCR检测方法至少包括以下步骤：
(1)提取模板DNA；
(2)将所述数字PCR检测试剂与引物、探针及DNA模板混合，配制微滴式数字PCR反应混合液
(3)将所述微滴式数字PCR反应混合液和微滴生成油加入微滴生成卡中，与微滴生成仪中生成微滴，转移到96孔反应板中进行PCR反应；
(4)将96孔板置于微滴读取仪中，利用软件直接读取结果，进行分析，计算HBV总拷贝数和突变拷贝数，计算突变比例。
本发明所述的微滴式数字PCR扩增反应条件为：95℃作用10分钟，然后94℃作用30秒、58℃作用45秒，共进行40个循环，最后98℃作用10分钟灭活，降温至4℃。
与现有技术相比，本发明具有以下的优点和效果：
(1)本发明结合数字PCR技术和突变扩增阻滞系统方法，兼具数字PCR技术的灵敏度高、能够绝对定量及突变扩增阻滞系统方法检测突变特异性强的特点，在HBV发生216位点突变基因丰度很低的情况下进行特异性检测，能够精确快速得出低丰度突变的比例，可预警预测乙型肝炎重症化，为阻止ACLF发病和改善HBV感染者的预后提供依据。
(2)本发明基于数字PCR系统平台，具有绝对定量的优势，可直接根据结果计算得到突变比例。
(3)本发明检测方法操作简单，与传统荧光定量PCR技术的兼容性高，检测结果一致性好，具有较高的实际应用价值。
附图说明
图1：突变型为阴性，野生型为阳性的检测图。
图2：突变质粒模板与野生质粒模板比例为1:1000的检测图。
具体实施方式
通过下面具体的实例可以更容易的了解本发明的内容，本发明的保护范围不局限于下述特定的具体实施方案。
实施例一：
利用突变扩增系统(ARMS)方法设计合成检测HBVT216C突变的引物和探针
针对HBV216位点的突变，根据ARMS方法原理，利用Beacon Designer生物软件设计引物和探针，利用引物设计规则，手动输入含突变位点的上游引物，将突变位点放在上游引物3’端，软件搜索匹配的下游引物和探针；得到SEQ NO:1所示的正向引物、由SEQ ID:2所示的反向引物、由SEQ ID NO:3所示的探针，探针荧光基团为FAM，淬灭基团为BHQ1。
针对HBV保守序列，根据ARMS方法原理，利用Beacon Designer生物软件设计引物和探针，得到SEQ NO:4所示的正向引物、由SEQ ID:5所示的反向引物、由SEQ ID NO:6所示的探针，探针荧光基团为VIC，淬灭基团为BHQ1。
本发明的引物和探针是从多条引物和探针中筛选出最优化的，具有特异性强，灵敏度高的特性。
实施例二
HBV基因216位点的突变检测
1.准备检测样本：将含有HBV突变型位点基因(T216C)的质粒和含有HBV保守序列的质粒 按照拷贝数含量为1:100、1:1000和1:5000的比例混合，取2ul混合后的DNA检测。
2.配制ddPCR反应液：2*ddPCR supermix for probes(no dUTP)(BIO-RAD)，用于检测216突变位点的上下游引物和探针，用于检测HBV保守序列的上下游引物和探针，待测混合型DNA模板，蒸馏水补充至总体积为20ul，配制ddPCR反应液，其中两对上下游引物的浓度均为500nM,两个探针的含量为250nM。
3.将配制好的20μlPCR反应液，转移至微滴发生卡中，加入70μl微滴发生油后，利用微滴生成仪制备反应微滴。
4.将生成的微滴全部转移至96孔PCR反应板，于普通PCR仪上进行扩增，扩增程序如下：
Figure PCTCN2020136277-appb-000001
5.将扩增后的96孔板放入微滴读取仪中，并在软件QuantaSoft上设定检测模式，检测FAM和VIC的荧光信号，仪器会自动分析每个微滴中荧光信号，根据检测得出的突变型HBV绝对定量，计算样品中HBV突变型占总的HBV含量的比率。

Claims (8)

  1. 一种基于微滴式数字PCR技术的乙型肝炎病毒T216C突变检测的方法，其特征在于，所述方法包括如下步骤：
    (1)对HBV基因的T216C突变位点设计PCR扩增引物和探针；
    (2)对HBV基因的保守序列设计PCR扩增引物和探针；
    (3)对步骤(1)和(2)的突变位点基因和保守序列基因进行两通道的微滴式数字PCR检测；
    (4)通过对HBV突变位点的微滴式数字PCR扩增结果确定待测样品是否存在突变及突变拷贝数，通过对HBV基因保守序列基因的数字PCR扩增结果确定样品中HBV基因总数，通过比较来计算判断HBV突变基因占HBV基因总数的比例。
  2. 根据权利要求1所述的方法，其特征在于，所述用于步骤(1)检测HBV基因T216C突变位点的引物和探针包括由SEQ NO:1所示的正向引物、由SEQ ID:2所示的反向引物、由SEQ ID NO:3所示的探针。
  3. 根据权利要求1所述的方法，其特征在于，所述用于步骤(2)检测HBV基因保守序列的引物和探针包括由SEQ NO:4示的正向引物、由SEQ ID:5所示的反向引物、由SEQ ID NO:6所示的探针。
  4. 根据权利要求2或3所述的方法，其特征在于，所述检测探针5’端标记荧光染料，标记荧光基团为FAM、VIC或HEX中的任意一种；3’端标记淬灭基团，所述淬灭基团为BHQ1、BHQ2或MGB中的任意一种。
  5. 根据权利要求4所述的方法，其特征在于，所述标记荧光基团VIC荧光；所述淬灭基团为BHQ1。
  6. 根据权利要求1所述的方法，其特征在于，所述用于步骤(3)两通道检测的数字PCR检测包括检测试剂，还包括微滴生成卡，96孔反应板、铝箔热封膜和读数油。
  7. 根据权利要求6所述的方法，其特征在于，步骤(3)包括以下步骤：
    1)提取模板DNA；
    2)将所述数字PCR检测试剂与引物、探针及DNA模板混合，配制微滴式数字PCR反应混合液；
    3)将所述微滴式数字PCR反应混合液和微滴生成油加入微滴生成卡中，与微滴生成仪中生成微滴，转移到96孔反应板中进行PCR反应；
    4)将96孔板置于微滴读取仪中，利用软件直接读取结果，进行分析，计算HBV总拷 贝数和突变拷贝数，计算突变比例。
  8. 根据权利要求7所述的方法，其特征在于，所述的微滴式数字PCR扩增反应条件为：95℃作用5分钟，然后95℃作用15秒、58℃作用30秒，共进行40个循环，最后98℃作用10分钟灭活，降温至4℃。
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