基于丙型肝炎病毒NS5B区的巢式RT-PCR和基因分型的比较研究  

A comparison of nested RT-PCR targeting the NS5B region and genotyping of the hepatitis C virus

作　　者：尹琦[1,2] 杨庆利[1] 方钟燎[1] 王学燕[1] 李海[1] 陈钦艳[1] 谭超[1] 罗洲[2] 

机构地区：[1]广西壮族自治区疾病预防控制中心广西病毒性肝炎防治研究重点实验室,广西南宁530028 [2]柳州市柳铁中心医院南方医科大学附属柳州医院,广西柳州545007

出　　处：《中国病原生物学杂志》2016年第8期685-689,695,共6页Journal of Pathogen Biology

摘　　要：目的研究不同引物组合和病毒载量对丙型肝炎病毒（HCV）RNA逆转录和NS5B区巢式PCR反应效率,以及由于逆转录引物不同对基因分型结果的影响。方法用荧光定量PCR检测病毒载量。分别用Oligo（dT）15和随机引物pd（N）6将HCV RNA逆转录合成cDNA,用针对5’-NTR区的巢式PCR进行检测;分别用针对NS5B区的简单引物和简并引物组合进行巢式PCR,比较各组扩增效率。对NS5B区第2轮PCR产物测序并比较基因分型结果的一致性。结果用Oligo（dT）15引物逆转录和随机引物逆转录合成cDNA,5’-NTR区扩增的阳性率分别为87.5%和98.4%,差异有统计学意义（χ2=17.6,P〈0.001）。用HCV1/HCV2-122S/123A简单引物组合和ENO2/ENO4-NS5S3/NS5A5简并引物组合对Oligo（dT）15逆转录cDNA产物进行NS5B区的巢式PCR扩增,阳性率分别为11.5%和35.4%,差异有统计学意义（χ2=30.7,P〈0.001）;用上述两组引物组合对随机引物逆转录cDNA进行NS5B区巢式PCR扩增,其阳性率分别为45.3%和94.3%,差异有统计学意义（χ2=109.1,P〈0.001）。随机引物在不同载量病毒组中的逆转录反应阳性率差异无统计学意义（χ2=2.00,P〉0.05）;随机引物配合简并引物组合在不同载量病毒组NS5B区扩增的阳性率差异无统计学意义（χ2=5.18,P〉0.05）。经Oligo（dT）15引物和随机引物合成cDNA,用简并引物组合对NS5B区进行巢式PCR扩增,通过核酸测序确定HCV基因型完全一致。结论使用pd（N）6随机引物进行逆转录,结合ENO2/ENO4-NS5S3/NS5A5简并引物组合可实现对不同载量HCV的基于NS5B区的高效巢式PCR扩增和基因分型。Objectives To determine the efficiency of reverse transcription of hepatitis C virus（HCV）RNA and nested PCR targeting the NS5 Bregion with different primer combinations and samples with different viral loads.To compare the consistency of genotyping,which may be influenced by reverse transcription with different primers. Methods The viral load of HCV samples was detected with real-time RT-PCR and classified as low（〈 104 IU/mL）,middle（104 106 IU/mL）,or high（〉106 IU/mL）.cDNA was synthesized by reverse transcription using the primers Oligo（dT）15and pd（N）6and verified with nested PCR targeting the 5’-NTR.The NS5 Bregion was amplified with nested PCR using the simple primers HCV1,HCV2-122 S,and 123 Aand the degenerate primers ENO2,ENO4-NS5S3,and NS5A5.DNA products from the 2nd round of PCR were sequenced in order to genotype the virus and construct a genetic evolutionary tree.The amplification efficiency of nested RT-PCR and its consistency with genotyping were also analyzed. Results cDNA was reverse transcribed using the primer Oligo（dT）15or a random primer,and the 5’-NTR was amplified with nested PCR.When the primer Oligo（dT）15was used,87.5% of samples tested positive for the target region;when a random primer was used,98.4% of samples tested positive for the target region（χ2=17.6,P〈0.001）.cDNA was reverse transcribed using the primer Oligo（dT）15along with the simple primers or degenerate primers,and the NS5 Bregion was amplified with nested PCR.When the primer Oligo（dT）15and simple primers were used,11.5% of the PCR products tested positive for the target region;when primer Oligo（dT）15and degenerate primers were used,35.4% of the PCR products tested positive for the target region（χ2=30.7,P〈0.001）.cDNA was reverse transcribed using the primer Oligo（dT）15or pd（N）6and random primers,and the NS5 Bregion was amplified with nested PCR.When the primer Oligo（dT）15and random primers were used,45.3% of the PCR products tested positive for the target

关 键 词：丙型肝炎病毒 NS5B区 RT-PCR 病毒载量 

分 类 号：R373.21[医药卫生—病原生物学]