蚊类携带甲病毒属病毒CODEHOP RT-PCR检测方法的建立

    马思杰，胡群

　　大榭出入境检验检疫局，浙江 宁波 315812

　　摘要：目的  建立蚊类感染甲病毒属病毒的CODEHOP RT-PCR检测方法。方法 依据甲病毒属病毒非结构蛋白氨基酸序列，设计1对CODEHOP引物，建立甲病毒属病毒一步RT-PCR检测方法，分析该引物在对不同种甲病毒基因序列上的结合位点及所能获得的产物序列大小；通过检测甲病毒属基孔肯亚病毒JC2012株，评价该方法的特异性和灵敏度；对JC2012扩增产物进行Blast同源性比对。结果 建立的CODEHOPRT-PCR方法可特异性扩增基孔肯亚病毒核酸，目的片段的大小与预期结果相符，核酸的最小检出量为56.7pg。经Blast比对，结果与Genebank 公布的CHIKVJC2012序列结果一致。同时从GenBank获得的22种甲病毒均存在CODEHOP引物结合位点，扩增产物大小在510~550bp之间。结论  建立的甲病毒CODEHOP RT-PCR检测方法敏感、特异，可用于蚊类甲病毒感染检测。

　　关键词：甲病毒；一致-简并杂合寡核苷酸引物；检测

　　中图分类号：R373.9  文献标识码：B

　　Establishment of a CODEHOP RT-PCR method for detecting

　　Alphavirus carried by mosquito

　　MA Si-jie， HU Qun

　　Daxie Entry-exit Inspection and Quarantine， Ningbo， Zhejiang315812， China

　　Abstract：  Objective  To develop a CODEHOP RT-PCRmethod for detecting Alphavirus carried by mosquito.

　　Methods  According to the amino acid sequences ofnonstructural protein of Alphavirus， a pair of CODEHOP primers wasdesigned. Based on which a method of one step RT-PCR wasestablished for detecting Alphavirus. The primer binding sites indifferent species of Alphavirus and the size of the products wereanalyzed. The sensitivity and specificity of the method wereevaluated according to the result of detecting CHIKV JC2012. Results  The CODEHOP RT-PCR method could amplify theCHIKV JC2012 specially， the sequence was completely consistent withthe published sequence of CHIKV JC2012 and the limit of detectionof nucleic acid was 56.7 pg. Meanwhile， all of the 22 Alphaviruscould be find the binding sites for the CODEHOP primers， and allthe amplifications were 510~550 bp.  Conclusion  TheCODEHOP RT-PCR method is specific and sensitive which could be usedto detect mosquito-brone Alphavirus.

　　Key words： Alphavirus； Consensus-degenerate hybridoligonucleotide primer （CODEHOP RT-PCR）； Detection

　　《中国国境卫生检疫杂志》2014年3期