巢式逆转录聚合酶链反应在轮状病毒型别鉴定中的适用性  

Applification of nested-RT-PCR for rotavirus genogype identification

作　　者：王革 胡广宏 王云瑾 马超 WANG Ge;HU Guang-hong;WANG Yun-jin;MA Chao(Department of Quality Control,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司质量检定室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2020年第2期28-32,共5页Progress In Microbiology and Immunology

摘　　要：目的以巢式逆转录聚合酶链反应(nested reverse transcription PCR, Nested-RT-PCR)鉴定轮状病毒型别,并对其适用性进行验证。方法提取轮状病毒基因组RNA,以逆转录聚合酶链反应(reverse transcription PCR, RT-PCR)扩增轮状病毒VP7基因序列,再以Nested-RT-PCR扩增各型别轮状病毒特异性目的基因片段,将扩增产物经琼脂糖凝胶电泳检测其基因片段的大小,并与Genbank报道的相应基因序列片段预计大小进行对比,以确定各毒株的型别。分析该方法的特异性、灵敏度及准确性,对Nested-RT-PCR鉴定轮状病毒型别的适用性进行验证。结果 Nested-RT-PCR法可特异性扩增各型别轮状病毒特异性目的基因片段,所检测的轮状病毒型别特异性目的基因片段与Genbank报道的相应基因序列片段大小相符,可用于轮状病毒型别鉴定。适用性验证结果显示,此方法所用引物特异性好,能特异性扩增相应型别的轮状病毒目的基因片段;具有较高的灵敏度,可检出RNA质量浓度为0.5 ng/μL的样本,其检测灵敏度高于PrimeScriptTM One Step RT-PCR试剂盒检测灵敏度;准确性检测结果与PrimeScriptTM One Step RT-PCR试剂盒检测结果一致,且同型别轮状病毒检测重复试验结果一致。结论该方法特异、灵敏、准确,可有效判断轮状病毒毒株型别,避免了PrimeScriptTM One Step RT-PCR试剂盒检测由于样本含量低而造成的检测结果的假阴性,适用于轮状病毒毒株的检测和多价轮状病毒疫苗的质量控制。Objective To identify the types of rotaviruses based on nested-RT-PCR and to evaluate its applicability. Methods Rotavirus genomic RNAs were extracted and reverse transcribed to amplify rotavirus VP7 gene, then specific genes fragments were amplified with nested PCR. The size of specific gene fragment was determined with agarose gel electrophoresis and compared with that of predicted from the corresponding sequence recorded in Genbank in identification the types of rotavirus. The specificity, sensitivity and accuracy of this method were tested and its applicability was evaluated. Results The specific gene fragments of rotavirus could be amplified by nested-RT-PCR, and the sizes of specific gene fragments from different types of rotavirus were similar to the targets in Genbank,as a consequence it could detect the type of rotavirus strain. The method was validated for its availability, and the results showed that it had a good specificity and amplified the specific gene fragments;it had a good sensitivity with a working concentration 0.5 ng/μL, better than that from the PrimeScriptTM One Step RT-PCR;the accuracy tests showed consistent with PrimeScriptTM One Step RT-PCR and the repeated tests were consistent with those from the same type of rotavirus. Conclusion The performance of this method showed it had satisfactory specifity, sensitivity and accuracy, which could be effectively used in identification the genotypes of rotaviruses and in avoidance false negatives caused by samples with insufficient content, thus it could be suitable in detection of rotavirus strain and quality control in preparation of rotavirus vaccine.

关 键 词：巢式逆转录聚合酶链反应 轮状病毒 型别鉴定 验证 

分 类 号：R392[医药卫生—免疫学]