4株蚊媒病毒多重RT-PCR快速检测方法的建立

Development of Multiplex RT-PCR Methods for Detection of Arboviruses

目的建立登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒的多重RT-PCR快速检测方法。方法参照病毒核酸序列设计多重RT-PCR引物,并检索GenBank国际基因序列数据库初步验证其特异性,随后对Mg2+、dNTP及引物浓度,RT-PCR反应条件进行优化,建立稳定、特异的多重RT-PCR快速检测4株病毒方法 ,并以同属于黄病毒科的登革3型病毒、登革4型病毒及甲病毒属基孔肯亚病毒、辛德毕斯病毒为对照,验证其特异性。结果应用多重RT-PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;采用多重RT-PCR引物对登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒进行扩增,分别获得574、251、879、422 bp片段,与设计相符,而对照病毒组均无非特异性扩增条带。结论实验证明,所建立的多重RT-PCR方法能够快速地检测登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒,为其检测提供了一种方便易行的方法 。

Objective To develop a multiplex reverse transcription-polymerase chain reaction（RT-PCR） method for the detection of dengue virus type 1 and 2,Japanese encephalitis virus and Yellow Fever virus.Methods Based on the genomes sequence analysis of these viruses,four pair of primers were designed,the specificity of the primers was checked by comparing with the GenBank DNA sequence database.Then the optimal reaction conditions of the multiplex RT-PCR were established.The specificity of the RT-PCR was tested using the homologous virus,namely the dengue virus type 3 and 4,Sindbis virus and Chikungunya virus.Results Fragments of 574,251,879 and 422 bp,corresponding to the dengue virus type 1,2,Japanese encephalitis virus and Yellow Fever virus,respectively,were seen in the multiplex RT-PCR reaction.Amplification was not seen in the control group.Conclusion Multiplex RT-PCR can be used in the rapid detection of dengue virus type 1,2,Japanese encephalitis virus and Yellow Fever virus.