摘要
目的 建立适用于检测人用猪源性生物制品中外源性乙脑病毒(JEV)的嵌套式RT- PCR方法。方法 根据已公布的JEV序列(GenBank登录号为M5 5 5 0 6 ) ,设计合成两对引物,对JEV感染的乳鼠脑组织和培养JEV的BHK 2 1细胞抽提RNA进行RT PCR和RT nestedPCR ,将PCR产物进行了克隆测序;同时对猪细小病毒(PPV)、猴空泡病毒4 0 (SV4 0 )及正常乳鼠脑组织、正常BHK 2 1细胞、PK15细胞、BSC 1细胞提取核酸进行PCR ,并对2 10份临床样品进行了检测。结果 PCR产物经琼脂糖凝胶电泳检测,JEV感染的乳鼠脑组织和培养JEV的BHK 2 1细胞均扩增出10 15bp和6 2 2bp目的基因片段,而PPV、SV4 0及未感染JEV的乳鼠脑组织、正常BHK 2 1细胞、PK15细胞、BSC 1细胞均未见特异性扩增条带,2 10份猪组织样品中未检出阳性样品。RT nestedPCR检测的最低限度为10PFU病毒。从样品核酸的提取到PCR扩增及检测结果的报告可在8小时内完成。结论 本研究建立的RT NestedPCR方法具有快速、特异、敏感、可靠的特点,可用于人用猪源性生物制品中外源性JEV的污染检测。
To establish the reverse transcription-nested polymerase chain reaction (RT-nested PCR) method for detecting Japanese encephalitis virus (JEV) in human biological products of porcine origin. Two pairs of primers were designed according to the complete genomic RNA sequence of JEV (GenBank accession number M55506). RNA was extracted from suckling mice brain infected with JEV and BHK-21 cell inoculated with JEV. The specific amplified fragments of E gene which encoding envelope glycoprotein of JEV were cloned into the pGEM-T easy vector and transformed into E.coli DH5. The recombinant plasmid were identified by electrophoresis, PCR, and restriction endonuclease analysis, and confirmed by sequencing. Porcine parvovirus (PPV), simian virus 40 (SV40), BHK-21 cell, PK15 cell, BSC-1cell, normal suckling mice brain (JEV free), and 210 clinical swine tissues specimens were submitted to detect JEV by PCR. All PCR products were detected by agarose gel electrophoresis. JEV RT-PCR amplification product of the external primers was 1 015 bp, and RT-nested PCR amplification product of the internal primers was 622 bp. Non-JEV specimens had not shown those specific amplification fragments. Using internal primers in a nested RT-PCR, the sensitivity was increased approximately 1000-fold compared to that of the RT-PCR. The procedure including RNA extraction, amplification, and detection could be finished within 8 h. The results of the study demonstrated that the RT-nested PCR established here was a rapid, specific, sensitive, and reliable method for the detection of JEV in human biological products of porcine origin.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2005年第4期279-282,共4页
Chinese Journal of Zoonoses
基金
(8 63计划 )生物技术产品的质量标准和检测技术平台的研究(NO .2 0 0 3AA2Z3 481)
关键词
乙脑病毒
反转录-嵌套式聚合酶链反应
检测
Japanese encephalitis virus (JEV)
reverse transcription-nested polymerase chain reaction (RT-nested PCR)
detection
