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目的:建立乙脑病毒RT-PCR检测方法。方法:参照已发表的乙脑病毒全基因组序列,针对其衣壳蛋白C基因设计1对引物,提取乙脑病毒的RNA,逆转录为cDNA,进行PCR扩增,对检测方法进行优化,验证该方法的特异性及敏感性,并对90份临床样本进行检测。结果:所建立的乙脑病毒RT-PCR检测方法特异性和敏感性良好,以含乙脑病毒衣壳蛋白C基因的重组质粒DNA为模板,最低可检出10pgDNA。90份样本经RT-PCR检测,有23份扩增出特异性的目的条带。从小型猪脑组织中能分离鉴定出乙脑病毒。结论:已建立了乙脑病毒逆转录PCR检测方法,为乙脑病毒检测、流行病学调查奠定了基础。
Objective: To establish JE virus RT-PCR detection method. Methods: According to the published whole genome sequence of JE virus, a pair of primers was designed according to its capsid protein C gene. RNA of JE virus was extracted and reverse transcribed into cDNA for PCR amplification. The detection method was optimized and verified Method specificity and sensitivity, and 90 clinical samples were tested. Results: The specificity and sensitivity of the established JEV RT-PCR assay were good. The recombinant plasmid DNA containing the capsid protein C of JEV as a template could detect 10 pg DNA. Totally 90 samples were detected by RT-PCR, and 23 were amplified specific bands. Encephalitis virus can be isolated and identified from miniature porcine brain tissue. Conclusion: JE virus reverse transcription polymerase chain reaction (RT-PCR) detection method has been established and laid the foundation for the detection of JE virus and epidemiological investigation.