CN110878378A - 用于检测体液中戊型肝炎病毒的引物组合及其应用 - Google Patents
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Abstract
本发明公开了一种用于检测体液中戊型肝炎病毒的引物组合，其包括逆转录引物组、用于检测348bp片段引物组、用于检测B片段引物组；该方法旨在将微量的RNA通过逆转录预扩增得到较高浓度的DNA模板，结合琼脂糖凝胶电泳法检测样本中348bp片段与B片段情况；可检测样本RNA浓度低至100拷贝，且可以同时检测HEV基因348bp与B片段，本发明提供的检测方法灵敏度高，特异性强，操作简便，检测时间短，适用于大规模样品的快速检测，为传染源调查、传播环境、病毒来源工作奠定基础。
Description
技术领域
本发明属于生物检测技术领域，涉及一种用于体液中戊型肝炎病毒检测的引物组及其在传染源调查、传播环境、病毒来源相关方面的应用。
背景技术
戊型肝炎病毒（Hepatitis E virus，HEV）是一种严重危害人类健康的病毒性肝炎病原体，其发病率居人急性病毒性肝炎之首（Aggarwal R，et al, 2011）。世界卫生组织（WHO）数据显示全球每年约有2000万人感染HEV，其中约7万人死于HEV感染引起的病毒性肝炎（Rein, D.B., et al, 2012）。HEV是一种准包膜的单股正链RNA病毒（Emerson SU，etal, 2003）。HEV病毒颗粒直径约为27-34nm，基因组全长约7.2 kb，包含5’端及3’端的非编码区域（UTR）和三个开放阅读框（open reading frames；ORFs: ORF1、ORF2和ORF3）（TamAW，et al,1991）。其中ORF1主要编码与HEV复制相关的酶，包括甲基转移酶（MET）、解旋酶（HEL）、半胱氨酸蛋白酶（PCP）和RNA依赖的RNA聚合酶（RdRp）等（Agrawal S，et al, 2000）。另外还有一个高变区（V）和一个功能未知的Y区。ORF2主要编码HEV的衣壳蛋白，衣壳蛋白通过与病毒RNA 5’端的包装信号序列结合进行病毒颗粒的装配（Meng XJ 2010）。ORF3与ORF2部分相互重叠，且重叠的部分高度保守。目前ORF3蛋白功能还不清楚，有研究报道其可能与HEV的组装和释放有关。
目前，戊型肝炎的诊断方法很多，例如病毒的分离鉴定、酶联免疫吸附试验、聚合酶链式反应等。以核酸扩增技术为基础的检测方法是检测急性戊型肝炎病毒(HEV)感染的主要方法，但检测灵敏度差异很大。世界卫生组织（WHO）数据显示，来自10个国家的24个实验室采用常规HEV RNA检测方法，实验结果差距显著。目前，常规检测方法为检测HEV 348bp片段，测序时需要以348片段为基础，但其引物为一种兼并引物，检测范围窄，假阳性率高。并且，由于戊型肝炎病毒基因类型比较复杂，在应用PCR方法时，引物设计难度较大，包括引物探针的序列设计与选择、引物/探针用量、镁离子用量、反应条件和阴/阳性对照品等多项技术参数尚缺乏成熟的技术信息，因而还没有形成适应范围大、操作简便、准确可靠的实时检测试剂盒，限制了该方法的推广和普及。
发明内容
为克服现有技术的不足，本发明提供了一种用于检测体液中戊型肝炎病毒的引物组合，该发明利用348片段和B片段同时检测的方法，对HEV各种基因型实现快速检测；具有特异性强、适应范围广、操作简便、检测结果准确可靠、检测时间短、灵敏度高等优点，解决了检测中灵敏度低、单一引物组检测范围窄、假阳性率高的问题，适合大规模样品的快速检测，为传染源、传播环境、病毒来源的调查等工作奠定基础，有利于临床使用和推广。
本发明用于检测体液中戊型肝炎病毒的引物组合包括逆转录引物组、用于检测348bp片段引物组、用于检测B片段引物组；其中逆转录引物组包括如SEQ ID NO:2（HEV-B2）、SEQ ID NO:6（HEV2）所示的核苷酸序列以及随机引物，用于检测B片段引物组包括如SEQ ID NO:1～SEQ ID NO:4（HEV-B1、HEV-B2、HEV-B3、HEV-B4）所示的核苷酸序列；
本发明中使用的用于检测348bp片段引物组为常规公知引物，但不局限于该组引物，其包括如SEQ ID NO:5～SEQ ID NO:8（HEV1、HEV2、HEV3、HEV4）所示的核苷酸序列。
本发明一个目的是提供一种包含上述引物组合的检测戊型肝炎病毒的检测试剂。
本发明另一目的是提供包含上述引物组合的检测戊型肝炎病毒的检测试剂盒，所述检测试剂盒组分还包括下列常规组分：Green Mix、DEPC-ddH2O、MgCl2等。
本发明用于检测体液中戊型肝炎病毒的引物组合的检测方法，具体步骤如下：
（1）逆转录
取待测样本总RNA 12μL作为模板，置于200μL的EP管中，加入检测引物HEV2、HEV-B2、随机引物（Oligo(dT)18 primer）各0.5μL，离心机混匀2-3s；70℃预变性10min，然后冰上静置2min，再加入Recombinant RNase Inhibitor0.5μL、dNTP2μL、Murine Leukemia Virus酶0.5μL、5×MLVBuffer4μL，离心机混匀2-3s；25℃，5min在mRNA 3′端添加polyA尾巴；42℃，逆转录1h；72℃，使酶失活；
4℃，∞。
（2）HEV 348bp片段的巢式PCR检测
采用常规公知引物进行扩增，本发明使用的引物包括如SEQ ID NO:5～SEQ ID NO:8（HEV1、HEV2、HEV3、HEV4）所示的核苷酸序列，但不局限于该组引物；扩增方法为常规方法；
HEV B片段检测
配置第一轮PCR反应总体系为20μL：其中Green mix 混合液10μL、上下游引物HEV-B1、HEV-B2各0.5μL、逆转录产物cDNA5μL、用水补充至20μL；PCR反应条件为：
95℃，预变性5min；
95℃，变性30s；
50℃，退火30s；
72℃，延伸1min；循环
至
35次；
72℃，10min
4℃，∞；
配置第二轮PCR反应总体系为20μL：其中Green mix 混合液10μL、上下游引物HEV-B3、HEV-B4各0.5μL、第一轮PCR产物5μL、用水补充至20μL；PCR反应条件为：
95℃，预变性5min；
95℃，变性30s；
50℃，退火30s；
72℃，延伸1min；循环
至
35次；
72℃，10min
4℃，∞；
（3）琼脂糖凝胶电泳检测
取终产物5μL进行电泳，DL2000 DNA Marker为对照，观察检测样品中是否含有348bp片段或B片段(774bp)。
本发明方法的待测样品为粪便悬液、精液、尿液或血清。
本发明优点和技术效果如下：（1）灵敏度高；（2）特异性识别HEV小片段；（3）适用于各种基因型的片段检测；（4）操作简便，检测时间短；（5）检测结果准确可靠；
本发明引物组合利用348片段和B片段同时检测的方法，对HEV各种基因型均能实现快速检测，使用本发明方法可实现对体液样本中戊型肝炎病毒进行高效、准确的检测，且具有灵敏度高、特异性强、适应范围广、操作简便、检测结果准确可靠、检测时间短等优点，解决了灵敏度低的问题，适合大规模样品的快速检测，其对于临床戊肝早期筛查，指导临床用药，以及为传染源调查、传播环境、病毒来源工作奠定基础。
附图说明
图1是本发明引物组合对72例样品348bp片段检测的结果；
图2是本发明引物组合对72例样品B片段检测的结果；
图3是本发明针对不同浓度梯度样品的扩增结果。
具体实施方式
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍。显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，按照以下实施例，不难根据具体情况略作修改和变换而成功实施本发明，这些修改和变换均落在本申请权利要求的范围内。
实施例1：72例样品中348bp检测
1、实验材料
HEV1、HEV2、HEV3、HEV4、HEV-B2、dNTP、Recombinant RNase Inhibitor、MurineLeukemia Virus酶、5×MLVBuffer、DEPC-ddH2O、Green mix、1%的琼脂糖凝胶和电泳仪。
2、实验步骤及结果
2.1样品RNA提取
（1）取72个待测样品的血清各200μL，置于1.5mL EP管中，分别加入800μL RNAiso，漩涡震荡使其混合均匀，冰上静置5min；
（2）加入200μL氯仿，冰上静置15min，12000rpm离心15min；
（3）取离心后的上清液，置于1.5mL EP管中，加入上清液等体积的异丙醇，然后加入上清液体积1/10的冰乙酸，上下颠倒混合均匀；
（4）-20℃冰箱静置20min后，12000rpm离心15min；
（5）弃上清液，加入1mL无水乙醇，7500rpm离心5min；
（6）弃去无水乙醇后倒扣晾干；
（7）最后加入40μL DEPC水，吹打均匀。
2.2逆转录
（1）取提取的RNA样品12μL（剩余RNA加1ml无水乙醇，-40℃冰箱保存），HEV2、HEV-B2、随机引物（Oligo(dT)18 primer）各0.5μL，离心机混匀，70℃预变性10min，冰上静置2min；
（2）加入Recombinant RNase Inhibitor0.5μL、dNTP2μL、Murine Leukemia Virus酶0.5μL、5×MLV Buffer4μL，离心机混匀；PCR反应条件为：
25℃5min，在mRNA 3′端添加polyA尾巴；
42℃，逆转录1h；
72℃，使酶灭活10min；
4℃，∞；
2.3巢式PCR检测348bp片段
（1）第一轮PCR反应体系配置如下:Green mix 混合液10μL、上下游引物HEV1、HEV2各0.5μL、逆转录产物cDNA5μL、用水补充至20μL；PCR反应条件为：
95℃，预变性5min；
95℃，变性30s；
42℃，退火30s；
72℃，延伸30s；循环
至
35次；
72℃，10min
4℃，∞；
（2）第二轮PCR反应体系配置如下:Green mix 混合液10μL、上下游引物HEV3、HEV4各0.5μL、第一轮PCR产物5μL、用水补充至20μL；PCR反应条件为：
95℃，预变性5min；
95℃，变性30s；
42℃，退火30s；
72℃，延伸30s；循环
至
35次；
72℃，10min
4℃，∞；
2.4琼脂糖凝胶电泳
取PCR扩增产物5μL进行电泳，2000Marker为对照，Marker条带由上至下依次为2000bp、1000bp、750bp、500bp、250bp、100bp；样品中含有348bp片段的样品判定为阳性，结果见图1。
实施例2：72个样品中B片段的检测
1、实验材料
HEV-B1、HEV-B2、HEV-B3、HEV-B4、HEV2、dNTP、Recombinant RNase Inhibitor、MurineLeukemia Virus酶、5×MLV Buffer、DEPC-ddH2O、Green mix、1%的琼脂糖凝胶和电泳仪；
2、实验步骤及结果
2.1 RNA提取
（1）取72个待测样品的血清各200μL，置于1.5mlEP管中，分别加入800μL RNAiso，漩涡震荡使其混合均匀，冰上静置5min；
（2）加入200μL氯仿，冰上静置15min，12000rpm离心15min；
（3）取离心后的上清液，置于1.5mlEP管中，加入上清液等体积的异丙醇，然后加入上清液体积1/10的冰乙酸，上下颠倒混合均匀；
（4）-20℃冰箱静置20min后，12000rpm离心15min；
（5）弃上清液，加入1mL无水乙醇，7500rpm离心5min；
（6）弃去无水乙醇后倒扣晾干；
（7）最后加入40μL DEPC水，吹打均匀。
2.2逆转录
（1）取提取的RNA样品12μL，HEV2、HEV-B2、随机引物（Oligo(dT)18 primer）各0.5μL，离心机混匀，70℃预变性10min，冰上静置2min；
（2）加入Recombinant RNase Inhibitor0.5μL、dNTP2μL、Murine Leukemia Virus酶0.5μL、5×MLV Buffer4μL，离心机混匀；PCR反应条件为：
25℃，5min在mRNA 3′端添加polyA尾巴；
42℃，逆转录1h；
72℃，灭活10min；
4℃，∞；
2.3巢式PCR检测B片段
（1）第一轮PCR反应体系配置如下:Green mix 混合液10μL、上下游引物HEV-B1、HEV-B2各0.5μL、逆转录产物cDNA5μL、用水补充至20μL；PCR反应条件为：
95℃，预变性5min；
95℃，变性30s；
50℃，退火30s；
72℃，延伸1min；循环
至
35次；
72℃，10min
4℃，∞；
（2）第二轮PCR反应总体系配置如下：其中Green mix 混合液10μL、上下游引物HEV-B3、HEV-B4各0.5μL、第一轮PCR产物5μL、用水补充至20μL；PCR反应条件为：
95℃，预变性5min；
95℃，变性30s；
50℃，退火30s；
72℃，延伸1min；循环
至
35次；
72℃，10min
4℃，∞；
2.4琼脂糖凝胶电泳
取PCR扩增产物5μL进行电泳，2000Marker为对照，Marker条带由上至下以此为2000bp、1000bp、750bp、500bp、250bp、100bp；样品中含有774bp片段的样品判定为阳性，结果见图2。
根据实施例1、2的结果，实验结果统计如下；待测样本共72份，其中348bp呈阳性的共有23例，B片段呈阳性的共有9例，348bp片段与B片段同时呈阳性的共有4例，且检测结果与72样本临床诊断结果一致，从结果可以说明采用348片段和B片段同时检测诊断，相比于常规的348bp引物检测灵敏度更高，避免了假阳性结果的出现，提高了检测结果的准确率，本发明方法能够用于大规模样品的精准检测。
实施例3：不同浓度含348bp片段质粒、B片段质粒的PCR检测
1、实验材料
HEV-B1、HEV-B2、HEV-B3、HEV-B4、HEV1、HEV2、HEV3、HEV4、DEPC-ddH2O、Green mix、1%的琼脂糖凝胶和电泳仪
2、实验步骤及结果
2.1样品梯度稀释
将浓度为2.76×1010个/μL的含348bp片段和含B片段的PUC-HEV质粒液体，以10倍梯度稀释的方式，稀释至109-102拷贝，制备8个浓度的质粒液体，且同时以去离子水作为阴性对照；
2.2巢式PCR检测348bp片段，方法同实施例1中的2.3；
2.3巢式PCR检测B片段，方法同实施例2中的2.3；
2.4琼脂糖凝胶电泳
取PCR扩增产物5μL进行电泳，2000Marker为对照，Marker条带由上至下依次为2000bp、1000bp、750bp、500bp、250bp、100bp；结果见图3，从图3中可以看出348bp引物组合检测样本RNA浓度范围为104-106拷贝，而B片段引物组合能检测到低至102拷贝浓度的稀释样品，说明HEV 348bp片段和B片段结合检测相对于常规的348bp引物检测来说灵敏度更高。
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Claims (3)
1.一种用于检测体液中戊型肝炎病毒的引物组合，包括逆转录引物组、用于检测348bp片段引物组，其特征在于：还包括用于检测B片段引物组，用于检测B片段引物组包括如SEQID NO:1～SEQ ID NO:4所示的核苷酸序列，逆转录引物组包括如SEQ ID NO:2、SEQ ID NO:6所示的核苷酸序列以及随机引物。
2.权利要求1所述的用于检测体液中戊型肝炎病毒的引物组合在制备检测戊型肝炎病毒的检测试剂中的应用。
3.权利要求1所述的用于检测体液中戊型肝炎病毒的引物组合在制备检测戊型肝炎病毒的检测试剂盒中的应用。
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Application publication date: 20200313 |