WO2016123895A1 - 一种用于检测乙型肝炎病毒cccDNA的定性和绝对定量试剂盒 - Google Patents

一种用于检测乙型肝炎病毒cccDNA的定性和绝对定量试剂盒 Download PDF

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WO2016123895A1
WO2016123895A1 PCT/CN2015/081800 CN2015081800W WO2016123895A1 WO 2016123895 A1 WO2016123895 A1 WO 2016123895A1 CN 2015081800 W CN2015081800 W CN 2015081800W WO 2016123895 A1 WO2016123895 A1 WO 2016123895A1
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dna
cccdna
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廖勇
牟迪
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  • the invention relates to the field of bioengineering, in particular to a qualitative kit and an absolute quantitative kit for effectively detecting hepatitis B virus DNA and hepatitis B virus covalently closed circular DNA and a method thereof.
  • PCR Polymerase chain reaction
  • PCR is a semi-quantitative and qualitative method for endpoint analysis of amplified products based on agarose gel electrophoresis. Its main advantages are simplicity and low cost.
  • the commonly used real-time fluorescent quantitative PCR is the amount of reaction product accumulated by the accumulation of fluorescent signals during the reaction, but the quantitative quantitative PCR requires a standard reference, which is only a relative quantitative method, and the amplification efficiency is Changes also affect the results of real-time PCR.
  • Digital PCR Digital PCR, Digital PCR, is an absolute quantification technique for nucleic acid molecules that enables highly accurate quantification of DNA of interest. Digital PCR allows you to directly count the copy number of a DNA molecule of interest.
  • Digital PCR is based on the use of traditional PCR and fluorescent probes, and does not require the establishment of a standard curve, nor does it require a standard reference for absolute quantification of nucleotides.
  • the main principle is to disperse a PCR system into 20,000 water-in-oil droplets. After amplification by ordinary PCR, droplets with fluorescent signals are considered positive, and droplets without fluorescent signals are regarded as negative.
  • the Poisson distribution performs a statistical analysis of the positive and negative droplets to perform an absolutely accurate quantification of the DNA molecule of interest in the test sample.
  • Hepatitis B virus is a serious hazard to human health.
  • the main replication/living process in the human body is: hepatitis B virus (HBV) binds to liver surface receptors, and cytoplasmic deviral capsids are loosely incompletely closed.
  • HBV hepatitis B virus
  • the circular DNA enters the nucleus, and under the action of the host and the viral DNA polymerase, the negative strand DNA is used as a template to prolong the repair of the DNA fissure region, and the two ends of the positive strand are ligated to form a double-stranded cccDNA, and the cccDNA is transcribed into 4
  • the viral mRNA, mRNA is translated to form a viral protein and reverse transcribed to form single-stranded DNA, and then the negative strand DNA is used as a template to synthesize positive strand DNA by the action of viral DNA polymerase, and together with the negative strand DNA, a new rcDNA is formed.
  • the latter is then packaged by viral surface proteins (capsid proteins) to form infectious virus particles and released outside the cell.
  • cccDNA is the original template for RNA replication of hepatitis B virus pre-genomics. Although its content is small, there are only about 5 to 50 copies in each liver cell, but it is of great significance for the replication of hepatitis B virus and the establishment of infection status. Some nucleoside analog antiviral drugs can not effectively remove cccDNA. Once cccDNA is present in the body, HBV may replicate again, leading to recurrence of hepatitis B. Therefore, it is a key factor for the repeated infection of HBV and the relapse of antiviral drugs. Only by clearing the cccDNA in the nucleus can the virus carrying status of hepatitis B patients be completely eliminated, which is the target of antiviral therapy. Because of the low content of cccDNA in cells, detection is relatively difficult.
  • Nested polymerase chain reaction is also called nested PCR.
  • HBV cccDNA in serum by nested polymerase chain reaction (nPCR) [J]. Chinese Journal of Medical Laboratory, 2005, 6(3): 168-170.]
  • the HBV cccDNA standard control plasmid was detected by the method, and the results showed that 5 ⁇ 10 5 copies/L HBVcc cDNA could be detected by the method, but the two-step method was easy to produce errors during sampling, and it was easy to cause pollution, and it took a long time and was cumbersome.
  • the template DNA was subjected to 15 cycles of single-strand extension using the insert and primer.
  • the cccDNA was able to extend smoothly to form a nascent strand due to the integrity of the positive strand; and other forms of HBV DNA, such as rcDNA,
  • HBV DNA such as rcDNA
  • This method detects a positive result of liver tissue samples of 10 5 -10 6 copies/ml.
  • the problem is that if the first round of amplification products are used as a template without purification, the efficiency of the second round of amplification will be directly affected by the complexity of the composition, and purification will affect the accuracy.
  • Two probes are designed for the target DNA, one is called the initial probe, the other is called the invasive probe, and the first probe has one segment of the oligonucleotide sequence at the 5-terminal end that is not complementary to the target DNA, and the invading probe
  • the single base at the 3 terminus is not complementary to the target DNA
  • the flap endonuclease I cleaves the oligonucleotide sequence at the 5th end of the initial probe that is not complementary to the target DNA, and the oligonucleotide sequence has a luminescent
  • the group is combined with a fluorescence resonance energy transfer probe of the quenching group to generate a fluorescent signal.
  • Wong et al.6 designed an invasive probe that binds to the direct repeat sequence 2 of the upstream and negative strands of the positive strand. Since both the positive and negative strands of the cccDNA are intact, two fluorescent signals are generated, but the rc DNA is positive. The chain contains a gap, so only one kind of fluorescent signal can be generated, and cccDNA and rcDNA can be distinguished according to the fluorescent signal generated by the DNA.
  • the method has high specificity, the composition of the reaction system is complicated and the reaction efficiency is difficult to control. The required time is longer, except for the denaturation and hot start treatment, which needs to be circulated at 64 ° C for 240 min.
  • the Taq enzyme Under the guidance of the upstream primer, if the negative strand is intact, the Taq enzyme reaches the site to which the Taq-Man probe binds, and the probe is cleaved by its 5' ⁇ 3' exonuclease activity, thereby 3' end The quenching group loses its inhibitory effect on the 5'-emitting group and produces a fluorescent signal. Conversely, if the negative strand contains a gap, the chain extension due to the upstream primer cannot pass through the negative strand gap, so that no fluorescent signal can be produced. Thus, cccDNA and rcDNA are distinguished. The linear range of its quantitative detection is 1 x 10 2 to 1 x 10 7 copies/L.
  • This method compares and analyzes the sequences of 150 known AG subtypes in the HBV gene pool, and designs primers based on conserved regions to detect A, B, C, F, G genotypes, so the method can be used in 90% Asia Pacific. Detection of cccDNA in hepatitis B patients. This method is currently considered to be the gold standard for cccDNA quantification in clinical liver biopsy specimens, but this method is less specific.
  • the detection method of cccDNA mentioned in the patent is based on the real-time quantitative PCR detection method of HE et al., which is a quantitative probe for HBV negative strand gap design for real-time quantitative PCR detection of cccDNA, but in the case of this method
  • the rcDNA product is also produced, the specificity is poor, and the real-time quantitative PCR technique is used as a reference. It is only a relative quantitative technique, and it is not capable of absolute quantification, and the sensitivity is relatively low.
  • the qualitative detection of cccDNA is by Southern blot. This method is a classical method of molecular biology, but it has high technical requirements and low sensitivity, and it is not easy to popularize in clinical practice.
  • the present invention establishes a rapid and inexpensive qualitative detection which can be used for identifying the presence or absence of cccDNA by using DNase which is safe for closed circular DNA to remove non-cccDNA (including rcDNA, ssDNA, HBV DNA, etc.).
  • DNase which is safe for closed circular DNA to remove non-cccDNA (including rcDNA, ssDNA, HBV DNA, etc.).
  • a kit for qualitative and absolute quantitative detection of cccDNA with high specificity, high sensitivity, and simple and convenient operation.
  • the present invention provides a primer and a probe for detecting hepatitis C virus cccDNA, specifically, the DNA sequence is the upstream primer of SEQ ID NO. 1, and the DNA sequence is SEQ.
  • the downstream primer of ID NO. 2 and the DNA sequence is the Taqman probe of SEQ ID NO.
  • the present invention provides a kit for detecting hepatitis B virus cccDNA comprising:
  • the upstream primer DNA sequence is SEQ ID NO. 1: 5 'CTTCTCATCTGCCGGACC 3' (nt1561-1579)
  • the downstream primer DNA sequence is SEQ ID NO. 2: 5'CACAGCTTGGAGGCTTGA 3' (nt1865-1883)
  • the Taqman probe DNA sequence is SEQ ID NO. 3:
  • Primer design Primers were designed for the negative link of rcDNA.
  • Upstream primer 5'CTTCTCATCTGCCGGACC 3' (nt 1561-1579)
  • Downstream primer 5'CACAGCTTGGAGGCTTGA 3' (nt 1865-1883)
  • DNA was purified enzyme digestion (Plasmid-Safe TM ATP-Dependent DNase) closed circular DNA secure: The step (2) extracted DNA was treated with DNase Plasmid-Safe TM ATP-Dependent, effective degradation comprising The notched rcDNA and ssDNA have no effect on cccDNA, which can reduce non-specific amplification due to rcDNA, reduce rcDNA background content, and increase the specificity of the reaction. Digestion system: 3ugDNA, 5ul 10x buffer, 2ul 25m MATP, 10U Plasmid-Safe TM ATP-Dependent DNase, complement ddH 2 O to 50ul. The enzyme was inactivated by a constant temperature of 37 ° C for 30 min and a constant temperature of 70 ° C for 30 min.
  • the reaction system was: 1 uul of 10 uM upstream and downstream primers, 2 ul of 2x PCR reaction DNA polymerase, and 1 ul of DNA, supplemented with ddH 2 O to 20 ul.
  • Reaction conditions preheating at 95 ° C for 3 min, 95 ° C for 30 s, 58 ° C for 1 min, 72 ° C for 30 s, 35 cycles, 98 ° C for 10 min.
  • Primer and probe design Primers and probes were designed for the negative link of rcDNA.
  • Upstream primer 5'CTTCTCATCTGCCGGACC 3' (nt 1561-1579)
  • Downstream primer 5'CACAGCTTGGAGGCTTGA 3' (nt 1865-1883)
  • the Taqman probe DNA sequence is SEQ ID NO. 3:
  • DNA was purified enzyme digestion closed circular DNA secure The step (2) DNA extracted from closed circular DNA with the enzyme DNA safe (Plasmid-Safe TM ATP-Dependent DNase) process, which can effectively degrade comprising The notched rcDNA and ssDNA have no effect on cccDNA, which can reduce non-specific amplification due to rcDNA, reduce rcDNA background content, and increase the specificity of the reaction.
  • Digestion system 3ugDNA, 5ul 10x buffer, 2ul 25mMATP, 10U secure closed circular DNA enzyme DNA (Plasmid-Safe TM ATP-Dependent DNase), complement ddH 2 O to 50ul. The enzyme was inactivated by a constant temperature of 37 ° C for 30 min and a constant temperature of 70 ° C for 30 min.
  • a digital PCR reaction was carried out using an EvaGreen fluorescent dye method or a probe method.
  • Sensitivity of digital PCR reaction The detection range of HBV plasmid DNA by digital PCR can reach 10 5 -10 1 copy/ul.
  • Figure 1 Schematic, probe design and schematic diagram of PCR for detecting cccDNA
  • FIG. 1 Schematic diagram of the HBV replication process and the role of DNase for the safety of covalent circular DNA
  • Figure 3 PCR map of different concentrations of HBV plasmid DNA; in Figure A is the EcoRI enzyme for HBV plasmid
  • B is HBV plasmid digested with EcoRI and then cccDNA safe DNase
  • Figure 5 A graph of sensitivity results for digital PCR detection.
  • HBV cccDNA is a completely closed circular DNA
  • HBV rcDNA is an incompletely closed circular DNA.
  • the sequence is as follows:
  • Upstream primer 5'CTTCTCATCTGCCGGACC 3' (nt 1561-1579)
  • Downstream primer 5'CACAGCTTGGAGGCTTGA 3' (nt 1865-1883)
  • Upstream primer 5'CTTCTCATCTGCCGGACC 3' (nt 1561-1579)
  • Downstream primer 5'CACAGCTTGGAGGCTTGA 3' (nt 1865-1883)
  • the pcDNA3.1-HBV1.3 plasmid was diluted to 10 7 -10 1 copy/ul.
  • the hepg2.215 cell DNA was diluted to 500 ng, 100 ng, 50 ng, 10 ng, 5 ng, 1 ng.
  • the enzyme digestion system 3ug DNA, 5ul 10x buffer, 2ul 25mMATP, 10U DNase, complement ddH 2 O to 50ul.
  • the enzyme was inactivated by a constant temperature of 37 ° C for 30 min and a constant temperature of 70 ° C for 30 min.
  • the reaction system was: 1 ul of 10 um upstream and downstream primers, and 10 ul of 2x PCR reaction DNA polymerase, supplementing ddH 2 O to 20 ul.
  • Reaction conditions preheating at 5 ° C for 3 min, 95 ° C for 30 s, 58 ° C for 1 min, 72 ° C for 30 s, 35 cycles, 98 ° C for 10 min.
  • PCR results for different concentrations of different concentrations of HBV DNA of plasmid DNA and cell hepg2.215 using this method may show cccDNA amplified, and after Plasmid-Safe TM ATP-Dependent DNase digestion, non-specific amplification significantly reduced, and after digested with EcoRI and then enzyme treated DNA cccDNA secure no PCR product, PCR amplification described in cccDNA Plasmid-safe TM ATP-Dependent DNase cccDNA amplification of specific play an important role.
  • Upstream primer 5'CTTCTCATCTGCCGGACC 3' (nt 1561-1579)
  • Downstream primer 5'CACAGCTTGGAGGCTTGA 3' (nt 1865-1883)
  • the pcDNA3.1-HBV1.3 plasmid was diluted to 10 5 -10 1 copy/ul.
  • the enzyme digestion system 3ug DNA, 5ul 10x buffer, 2ul 25mMATP, 10U DNase, complement ddH 2 O to 50ul.
  • the enzyme was inactivated by a constant temperature of 37 ° C for 30 min and a constant temperature of 70 ° C for 30 min.
  • the probe reaction system was: 1 uul of 10 uM upstream and downstream primers, 2 x digital PCR Supermix 10 ul, 10 uM fluorescent probe 0.5 ul, DNA 1 ul, and complemented with ddH 2 O to 20 ul.
  • the EvaGreen fluorescent dye reaction system is: 10 uM of upstream and downstream primers each 1 ul, 2 x digital PCR Supermix 10 ul, 20 x fluorescent probe 1 ul, DNA 1 ul, complement ddH 2 O to 20 ul.

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Abstract

提供了一种用于检测乙型肝炎病毒cccDNA的定性和绝对定量试剂盒，该试剂盒包括：HBV DNA的提取试剂；Plasmid-Safe TM ATP-Dependent DNase；DNA序列为SEQ ID NO.1的上游引物，DNA序列为SEQ ID NO.2的下游引物，以及DNA序列为SEQ ID NO.3的Taqman探针；EvaGreen荧光染料；普通PCR DNA聚合酶；数字PCR DNA聚合酶。

Description

一种用于检测乙型肝炎病毒cccDNA的定性和绝对定量试剂盒 技术领域
本发明涉及生物工程领域，尤其涉及一种有效检测乙型肝炎病毒DNA以及乙型肝炎病毒共价闭合环状DNA的定性试剂盒和绝对定量试剂盒及其方法。
背景技术
聚合酶链反应技术(polymerase chain reaction，简称PCR)又称体外酶促基因扩增。由Mullis发明于1983年，它以敏感，特异，快速的核酸分析技术而著称，是分子生物学技术的一项突破。其基本原理为，模拟于DNA的自然复制过程，引物按照碱基配对与DNA模板互补结合以后，在DNA多聚酶的作用下，按照碱基配对的原则(A、T，C、G)，从引物开始合成与模板DNA互补的DNA链。经变性，退火，延伸等一次循环，DNA链数量增加一倍。传统的PCR是基于琼脂糖凝胶电泳的方法对扩增产物进行终点分析的一种半定量、定性方法，其主要的优点是简便易行，而且成本低廉。在PCR定量技术中，常用的实时荧光定量PCR是通过反应过程中荧光信号的累积来反应产物的量，但是荧光定量PCR需要标准品做参照，只是一种相对定量的方法，而且扩增效率的改变也会影响荧光定量PCR的结果。而数字PCR即Digital PCR，它是一种核酸分子绝对定量技术，能够高敏感的对目的DNA进行精确定量。数字PCR可让你能够直接数出目的DNA分子的拷贝数。数字PCR是建立于传统的PCR和荧光探针的使用基础之上，且不需要建立标准曲线，也不需要标准品做参照就能够高灵敏的对核苷酸进行绝对定量。其主要原理是将一个PCR体系分散成20000个油包水的微滴，通过普通PCR的扩增后，具有荧光信号的微滴视为阳性，不具有荧光信号的微滴视为阴性，再通过泊松分布对阳性微滴和阴性微滴进行统计学分析从而对检测样品中目的DNA分子进行绝对精确定量。
乙型肝炎病毒严重危害人类健康，在人体内其主要的复制/生活过程为：乙型肝炎病毒(HBV)与肝表面受体结合，胞质内脱病毒衣壳，其松弛的不完全闭合的环状DNA(rcDNA)进入细胞核，在宿主和病毒DNA聚合酶作用下,以负链DNA为模板,延长修补DNA裂隙区,完成正链两端连接而形成双链完整的cccDNA，cccDNA转录形成4种病毒mRNA，mRNA翻译形成病毒蛋白并逆转录形成单链DNA，再以负链DNA作为模板,通过病毒DNA聚合酶的作用,合成正链DNA,与负链DNA一起组成新的rcDNA。后者再通过病毒表面蛋白(衣壳蛋白)包装形成具备感染能力的病毒颗粒并释放到细胞外。
可见cccDNA是乙肝病毒前基因组RNA复制的原始模板，虽然其含量较少，每个肝细胞内只有约5～50个拷贝，但对乙肝病毒的复制以及感染状态的建立具有十分重要的意义，现有的核苷类似物抗病毒药物并不能有效的清除cccDNA，体内一旦存在cccDNA，HBV就有可能再次复制，导致乙肝复发。因此其是HBV持续感染和抗病毒药物停用后病情反复的关键因素，只有清除了细胞核内的cccDNA，才能彻底消除乙肝患者病毒携带状态，是抗病毒治疗的目标。由于细胞内cccDNA含量较少，检测上相对困难。
国内外对于cccDNA定量检测的方法有很多，基本上都是利用cccDNA两条链均是完整的,而rcDNA存在缺口这个区别，通过荧光定量PCR来实现。
1、巢式聚合酶链反应也称套式PCR。
由于使用了两对引物并且进行了两轮扩增反应,第一对引物跨越环状HBV-DNA的DR1、DR2重复序列旁的两个缺刻，第二次PCR以一次PCR产物为模板扩增rcDNA缺口内部区域。其引物设计的原理也是基于cccDNA与rcDNA结构的不同，因此,cccDNA能够被扩增,而rcDNA则不能扩增。董庆鸣等[董庆鸣,魏红山,庄辉,等.套式聚合酶链反应法(nPCR)检测血清中HBV cccDNA[J].中国医学检验杂志,2005,6(3):168-170.]用该法检测HBV cccDNA标准对照质粒,结果表明该法可以检测到5×105拷贝/L HBVcccDNA，但是两步法在取样时容易产生误差，且容易产生污染，并且耗时较长，较繁琐。
2、嵌合引物两步法荧光定量PCR。
Shao等[SHAO JB,CHEN Z,NI WQ,et al.A quantitative method to detect HBV cccDNA by chimeric primer and real-time polymerase chain reaction[J].J Virol Methods,2003,112(1-2):45-52.]根据非cccDNA形式的HBV基因组存在缺口,设计由两段序列构成的嵌合引物。其3′端的12个碱基与正链(1604～1615)互补,结合位点为负链缺口上游DR2缺口下游,5′端的一段与人类免疫缺陷病毒的部分基因序列一致而与HBV基因组无同源性。首先用该嵌和引物对模板DNA进行15个循环的单链延伸,在该反应过程中,cccDNA因为正链完整,引物能够顺利延伸形成一条新生链；而HBV DNA的其他形式,如rcDNA,由于DR2区存在缺口,引物的延伸停止于DR2区,不能产生单链延伸产物。新生链形成以后,设计另一对引物,一个与嵌合性引物的片段B杂交,另一个与正链DR2后的序列互补,这样可以保证只能检测单链延伸反应的产物而不能直接检测HBV基因。此方法检测肝组织样本阳性结果为105-106拷贝/ml。但存在的问题是第一轮扩增产物如果不经过纯化直接充当模板，则会因为 成分复杂而直接影响第二轮扩增的效率，如果纯化则会影响准确性。
3、入侵检查
针对目标DNA设计两种探针，一种称为初始探针，另一种称为入侵探针，初始探针的5端有1段寡核苷酸序列不与目标DNA互补，入侵探针的3端的单个碱基不与目标DNA互补，flap核酸内切酶I将初始探针5端不与目标DNA互补的那段寡核苷酸序列剪切下来，此段寡核苷酸序列与具有发光基团和淬灭基团的荧光共振能量转移探针结合，从而产生荧光信号。Wong等6根据此原理，设计了与正链上游、负链下游HBV直接重复序列2区结合的入侵探针，由于cccDNA正链、负链均完整，故产生2种荧光信号，但rc DNA正链含有缺口，故只能产生1种荧光信号，可根据DNA产生的荧光信号区分cccDNA、rcDNA。此方法虽然特异性较高，但反应体系成分较复杂，反应效率难以控制。所需时间较长，除了变性、热启动处理还需在64℃条件下循环反应240min。
4、Light CyclerTM实时定量PCR方法。
Singh等[Singh M,Dicaire A,Wakil AE,Luscombe C,Sacks SK.Quantitation of hepatitis B virus(HBV)covalently closed circular DNA(cccDNA)in the liver of HBV-infected patients by LightCycler real-time PCR.J Virol Methods 2004；118:159-167.]研究的利用Light CyclerTM实时定量PCR仪检测HBV感染患者肝脏组织中cccDNA的方法。其引物设计是跨越了rcDNA的空隙区，并且采用双探针体系。其特点是为了消除rcDNA非特异性扩增对结果的干扰，除了设计特异性引物和探针外，还应用了Plasmid-SafeTM ATP-Dependent DNase降解rcDNA。该方法特异性较好，使用了Light CyclerTM实时定量PCR检测体系，检测结果相对稳定可靠，其敏感性为101拷贝/mg。但是该方法的探针合成、仪器设备及耗材成本均较高，限制临床应用。
5、实时定量PCR检测方法
He等[He ML,Wu J,Chen Y,Lin MC,Lau GK,Kung HF.A new and sensitive method for the quantification of HBV cccDNA by real-time PCR.Biochem Biophys Res Commun 2002；295:1102-1107.]根据cccDNA与rcDNA在结构和生化特性上的不同,建立了实时荧光定量PCR扩增法。他们将具有发光基团和淬灭基团的TaqMan探针设计于负链缺口的下游,与负链互补。在上游引物的引导下,若负链是完整的,Taq酶则到达Taq-Man探针所结合的位点,利用其5′→3′的外切酶活性将探针切断,从而3′端的淬灭基团失去对5′端发光基团的抑制作用,产生荧光信号。相反,若负链含有缺口,由于上游引物引发的 链延伸不能通过负链缺口,故不能产生荧光信号。这样,cccDNA和rcDNA得以区分开来。其定量检测的线性范围是1×102～1×107拷贝/L。该方法对比分析了HBV基因库中150条已知A-G基因亚型的序列,根据保守区域设计引物,能检测A、B、C、F、G基因型,故该方法可用于90％亚太地区的乙肝病人cccDNA的检测。目前认为该法是临床肝穿刺标本中cccDNA定量的金标准，但此方法特异性较低。
目前，有专利中提及的cccDNA的检测方法是基于HE等发明的实时定量PCR检测方法，此方法是针对HBV负链缺口设计的引物探针进行荧光定量PCR检测cccDNA，但在此方法情况下，也会产生rcDNA产物，特异性较差且荧光定量PCR技术去标准品作为参照，只是一个相对定量技术，并不能够进行绝对定量，灵敏度也相对较低。且目前对cccDNA进行定性检测是用Southern blot的方法，该方法是分子生物学的经典方法，但技术要求较高，敏感度低，在临床上也不易推广普及。
由此可知，上述定量方法都存在一定缺点，在临床应用中仍存在灵敏度较低，特异性差，成本较高，操作耗时久且不方便等问题。因此我们建立了一套既能用于鉴别cccDNA有无的快速、低廉的定性检测又能高特异性、高灵敏度且操作简单方便的对cccDNA进行绝对定量检测的试剂盒及方法，此方法是利用闭合环状DNA安全的DNA酶去除非cccDNA(包括rcDNA、ssDNA、HBV DNA等)，利用普通PCR操作快速、简单且价格低廉的特点对cccDNA进行定性检，利用探针法及EvaGreen荧光染料法通过数字PCR对cccDNA进行绝对定量检测，EvaGreen相比其他专利和文献中用到的SybrGreen对PCR扩增的抑制作用小，且不易产生非特异性扩增。
发明内容
针对以上不足，本发明建立了一套利用对闭合环状DNA安全的DNA酶去除非cccDNA(包括rcDNA、ssDNA、HBV DNA等)的既能用于鉴别cccDNA有无的快速、低廉的定性检测，又能高特异性、高灵敏度且操作简单方便的对cccDNA进行定性和绝对定量检测的试剂盒。
为了实现上述目的本发明采用的技术方案是：本发明提供了一种用于检测乙型肝炎病毒cccDNA的引物和探针，具体是DNA序列为SEQ ID NO.1的上游引物，DNA序列为SEQ ID NO.2的下游引物，以及DNA序列为SEQ ID NO.3的Taqman探针。
根据以上引物和探针，本发明提供了一种用于检测乙型肝炎病毒cccDNA的试剂盒包括：
(1)HBV DNA的提取试剂：细胞裂解液、Tris饱和酚(PH：7.6)、苯酚：氯仿：异戊醇＝25：24：1、无水乙醇、75％乙醇、TE缓冲液。
(2)闭合环状DNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)
(3)针对rcDNA负链缺口部位设计引物、探针。
上游引物DNA序列为SEQ ID NO.1：5'CTTCTCATCTGCCGGACC 3'(nt1561-1579)
下游引物DNA序列为SEQ ID NO.2：5'CACAGCTTGGAGGCTTGA 3'(nt1865-1883)
Taqman探针DNA序列为SEQ ID NO.3：
FAM-5`AGGCTGTAGGCATAAATTGGTCT 3'-BHQ(nt 1838-1861)
(4)EvaGreen荧光染料。
(5)普通PCR DNA聚合酶(2×Power Taq PCR MasterMix,百泰克)。
(6)数字PCR DNA聚合酶(Droplet PCR Supermix,BIO-RAD)。
利用对闭合环状DNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)cccDNA定性检测方法和cccDNA的数字PCR绝对定量检测方法：
1.cccDNA的普通PCR定性检测方法：
(1)引物设计：针对rcDNA负链缺口部位设计引物。
上游引物：5'CTTCTCATCTGCCGGACC 3'(nt 1561-1579)
下游引物：5'CACAGCTTGGAGGCTTGA 3'(nt 1865-1883)
(2)HBV DNA的提取：使用上述试剂盒中的HBV DNA的提取试剂，按常规方法进行DNA提取操作。
(3)闭合环状DNA安全的DNA酶酶切纯化(Plasmid-SafeTM ATP-Dependent DNase)：将步骤(2)中提取的DNA用Plasmid-SafeTM ATP-Dependent DNase处理，可有效的降解含有缺口的rcDNA和ssDNA，对cccDNA无影响，可减少由于rcDNA导致的非特异性扩增，降低rcDNA背景含量，提高反应的特异性。酶切体系：3ugDNA，5ul 10x buffer，2ul 25m MATP，10U Plasmid-SafeTM ATP-Dependent DNase，补足ddH2O至50ul。37℃恒温30min，70℃恒温30min使酶灭活。
(4)PCR反应
反应体系为：10uM的上下游引物各1ul，2xPCR反应DNA聚合酶10ul，DNA1ul，补足ddH2O至20ul。
反应条件：95℃预热3min，95℃30s，58℃1min，72℃30s,35个循环,98℃10min。
(5)琼脂糖凝胶电泳。
2.cccDNA的数字PCR绝对定量检测方法：
(1)引物、探针设计：针对rcDNA负链缺口部位设计引物、探针。
上游引物：5'CTTCTCATCTGCCGGACC 3'(nt 1561-1579)
下游引物：5'CACAGCTTGGAGGCTTGA 3'(nt 1865-1883)
Taqman探针DNA序列为SEQ ID NO.3：
FAM-5`AGGCTGTAGGCATAAATTGGTCT 3'-BHQ(nt 1838-1861)
(2)HBV DNA的提取：使用上述试剂盒中的HBV DNA的提取试剂，按常规方法进行DNA提取操作。
(3)闭合环状DNA安全的DNA酶酶切纯化：将步骤(2)中提取的DNA用闭合环状DNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)处理，可有效的降解含有缺口的rcDNA和ssDNA，对cccDNA无影响，可减少由于rcDNA导致的非特异性扩增，降低rcDNA背景含量，提高反应的特异性。酶切体系：3ugDNA，5ul 10x buffer，2ul 25mMATP，10U闭合环状DNA安全的DNA酶(Plasmid-Safe TM ATP-Dependent DNase)，补足ddH2O至50ul。37℃恒温30min，70℃恒温30min使酶灭活。
(4)利用EvaGreen荧光染料法或探针法进行数字PCR反应。
本发明的有益结果：
1、利用普通PCR对cccDNA进行定性检测，反应时间约70min左右，省时，方便，且成本低廉。而传统的Southern blot方法，一次检测需要数天时间，耗时长，步骤繁琐。本发明提供的试剂盒使临床快速判断药物治疗后cccDNA是否完全清除成为可能。
2、数字PCR反应的灵敏度：经数字PCR检测HBV质粒DNA其检测范围可达105-101copy/ul。
3、反应的特异性：用ECOR1酶切后加入闭合环状DNA安全的DNA酶后 无PCR产物。未加入此酶有PCR产物，说明加酶后PCR特异性都增高。
附图说明
图1：检测cccDNA的PCR的引物、探针设计及原理示意图；
图2：HBV复制过程及对共价环状DNA安全的DNA酶的作用原理图；
图3：HBV质粒DNA不同浓度的PCR图；图中A为HBV质粒用EcoRI酶
切后PCR结果；B为HBV质粒用EcoRI酶切再用cccDNA安全的DNA酶
处理后PCR结果；
图4；HepG2.215细胞DNA中cccDNA的PCR图；
图5：数字PCR检测灵敏性结果图。
具体实施方式
利用闭合环状DNA安全的DNA酶定性以及绝对定量检测cccDNA试剂盒包括(本试剂盒中未特别说明的试剂均可市购)：
HBV DNA的提取试剂：细胞裂解液、Tris饱和酚(PH：7.6)、苯酚：氯仿：异戊醇＝25：24：1、无水乙醇、75％乙醇、TE缓冲液。
Plasmid-SafeTM ATP-Dependent DNase：
有效的降解含有缺口的rcDNA和ssDNA，对cccDNA无影响，可减少由于rcDNA导致的非特异性扩增，降低rcDNA背景含量，提高反应的特异性，参见图2。
引物、探针：
都由生物公司合成，设计图如图1，HBV cccDNA为完全闭合环状DNA,而HBV rcDNA为不完全闭合环状DNA，针对HBV rcDNA负链缺口处设计的引物探针，以rcDNA为模板理论。序列如下：
上游引物：5'CTTCTCATCTGCCGGACC 3'(nt 1561-1579)
下游引物：5'CACAGCTTGGAGGCTTGA 3'(nt 1865-1883)
Taqman探针SEQ ID NO.3----
FAM-5`AGGCTGTAGGCATAAATTGGTCT 3'-BHQ(nt 1838-1861)
EvaGreen荧光染料
PCR产物：332bp
普通PCR反应所需的DNA聚合酶(2×Power Taq PCR MasterMix,百泰克)
数字PCR反应所需数字PCR Supermix(数字PCR操作步骤具体见BIO-RAD数字PCR操作说明)。
利用对闭合环状DNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)cccDNA定性检测方法和cccDNA的数字PCR绝对定量检测方法：
cccDNA的普通PCR定性检测方法：
1.1HBV DNA的提取：
(1)、150mmol/L NaCl,50mmol/L Tris-HCl(pH 7.4),10mmol/L EDTA,0.1％SDS蛋白酶K(800μg/mL)37℃过夜。
(2)、加入等体积tris饱和苯酚500上下颠倒充分混匀)后，离心(4℃12000r/min 10min)。
(3)、取上层水相500ul，再加入等体积500ul的(苯酚：氯仿：异戊醇＝25:24:1)，上下颠倒充分混匀，室温放置10min，离心(4℃12000r/min 10min)。
(4)、离心后上层水相450ul，加入4℃预冷的1/10体积的醋酸钠45ul(3mol/L，PH＝5.2)，再加入2倍体积的无水乙醇(4℃预冷)1.1ml，上下颠倒充分混匀，-20℃放置1h，再离心(4℃12000r/min 15min)。
(5)、去上清后加入70％乙醇1ml，洗涤2次(离心：4℃12000r/min 10min)
(6)、干燥、蒸发掉痕量的乙醇(约30min)，加入适量TE缓冲液40ul。冻存备用。
1.2引物的设计与合成：
都由生物公司合成，设计图如图1，序列：
上游引物：5'CTTCTCATCTGCCGGACC 3'(nt 1561-1579)
下游引物：5'CACAGCTTGGAGGCTTGA 3'(nt 1865-1883)
1.3cccDNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)酶切纯化：
将pcDNA3.1-HBV1.3质粒稀释成107-101copy/ul。将hepg2.215细胞DNA稀释成500ng，100ng，50ng，10ng，5ng，1ng。酶切体系：3ugDNA，5ul 10x buffer，2ul 25mMATP，10U DNA酶，补足ddH2O至50ul。37℃恒温30min，70℃恒温30min使酶灭活。
1.4PCR扩增:
反应体系为：10um的上、下游引物各1ul，2xPCR反应DNA聚合酶10ul，补足ddH2O至20ul。
反应条件：5℃预热3min，95℃30s，58℃1min，72℃30s,35个循环,98℃10min。
1.5琼脂糖凝胶电泳
分别用HBV质粒DNA和HepG2.215细胞，按照以上步骤进行操作，实验结果参见图2和图3。HBV质粒DNA不同浓度及hepg2.215细胞的不同浓度DNA的PCR结果:表明利用此方法可以扩增出cccDNA，且经过Plasmid-SafeTM ATP-Dependent DNase酶切后，非特异性扩增明显降低，且用EcoRI酶切再用cccDNA安全的DNA酶处理后无PCR产物，说明在cccDNA的PCR扩增中Plasmid-SafeTM ATP-Dependent DNase对cccDNA扩增的特异性起重要的作用。
利用闭合环状DNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)基于数字PCR绝对定量检测cccDNA方法：
2.1HBV DNA的提取：
(1)、150mmol/L NaCl,50mmol/L Tris-HCl(pH 7.4),10mmol/L EDTA,0.1％SDS蛋白酶K(800μg/mL)37℃过夜。
(2)、加入等体积tris饱和苯酚500上下颠倒充分混匀)后，离心(4℃12000r/min 10min)。
(3)、取上层水相500ul，再加入等体积500ul的(苯酚：氯仿：异戊醇＝25:24:1)，上下颠倒充分混匀，室温放置10min，离心(4℃12000r/min 10min)。
(4)、离心后上层水相450ul，加入4℃预冷的1/10体积的醋酸钠45ul(3mol/L，PH＝5.2)，再加入2倍体积的无水乙醇(4℃预冷)1.1ml，上下颠倒充分混匀，-20℃放置1h，再离心(4℃12000r/min 15min)。
(5)、去上清后加入70％乙醇1ml，洗涤2次(离心：4℃12000r/min 10min)
(6)、干燥、蒸发掉痕量的乙醇(约30min)，加入适量TE缓冲液40ul。冻存备用。
2.2引物、探针的设计与合成：
都由生物公司合成，设计图如图1，序列：
上游引物：5'CTTCTCATCTGCCGGACC 3'(nt 1561-1579)
下游引物：5'CACAGCTTGGAGGCTTGA 3'(nt 1865-1883)
FAM-5`AGGCTGTAGGCATAAATTGGTCT 3'-BHQ(nt 1838-1861)
PCR产物：332bp
2.3闭合环状DNA安全的DNA酶(Plasmid-SafeTM ATP-Dependent DNase)酶切纯化：
将pcDNA3.1-HBV1.3质粒稀释成105-101copy/ul。酶切体系：3ugDNA，5ul 10x buffer，2ul 25mMATP，10U DNA酶，补足ddH2O至50ul。37℃恒温30min，70℃恒温30min使酶灭活。
2.4PCR扩增:
探针法反应体系为：10uM的上下游引物各1ul，2x数字PCR Supermix10ul，10uM荧光探针0.5ul，DNA1ul，补足ddH2O至20ul。
EvaGreen荧光染料法反应体系为：10uM的上下游引物各1ul，2x数字PCR Supermix10ul，20x荧光探针1ul，DNA1ul，补足ddH2O至20ul。
反应条件：5℃预热3min，95℃30s，58℃1min，72℃30s,35个循环,98℃10min。(数字PCR操作步骤具体见BIO-RAD数字PCR操作说明)。结果如图5所示，利用数字PCR对cccDNA进行绝对定量，对cccDNA的检测下限可达到单个拷贝，说明此种绝对定量方法具有很高的灵敏度。图中的每个黑点代表一个拷贝，具体数值PCR反应仪器的数值处理系统会自动给出，此处未展示。

Claims (2)

  1. 一种用于检测乙型肝炎病毒cccDNA的引物和探针，其特征在于：包括DNA序列为SEQ ID NO.1的上游引物，DNA序列为SEQ ID NO.2的下游引物，以及DNA序列为SEQ ID NO.3的Taqman探针。
  2. 一种包含权利要求1所述引物和探针的用于检测乙型肝炎病毒cccDNA的定性和绝对定量试剂盒，其特征在于：包括，HBV DNA的提取试剂：细胞裂解液、Tris饱和酚、苯酚：氯仿：异戊醇＝25：24：1、无水乙醇、75％乙醇、TE缓冲液；
    Plasmid-SafeTM ATP-Dependent DNase；
    DNA序列为SEQ ID NO.1的上游引物，DNA序列为SEQ ID NO.2的下游引物，以及DNA序列为SEQ ID NO.3的Taqman探针；
    EvaGreen荧光染料；
    普通PCR DNA聚合酶；
    数字PCR DNA聚合酶。
PCT/CN2015/081800 2015-02-03 2015-06-18 一种用于检测乙型肝炎病毒cccDNA的定性和绝对定量试剂盒 WO2016123895A1 (zh)

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CN106520763A (zh) * 2016-12-02 2017-03-22 重庆医科大学附属第二医院 一种组合物、其应用以及含有该组合物的试剂盒
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CN106636466A (zh) * 2016-12-30 2017-05-10 南方医科大学南方医院 一种乙型肝炎病毒共价闭合环状dna精确定量的方法
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CN108285931B (zh) * 2018-03-30 2021-01-01 武汉大学 一种临床检测HBV cccDNA的微滴式数字PCR方法及试剂盒
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