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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CODEHOP RT-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56.7 pg。经Blast比对,结果与Genebank公布的CHIKV JC2012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510~550 bp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。
Objective To establish a CODEHOP RT-PCR method for detection of alphavirus in mosquitoes. Methods A pair of CODEHOP primers was designed according to the amino acid sequence of non-structural protein of alphavirus. One-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the alphavirus. The binding sites of the primers were analyzed. The specificity and sensitivity of this method were evaluated by detecting the methoviruses of Chikungunya strain JC2012. Blast homology comparison of JC2012 amplification products was carried out. Results The established CODEHOP RT-PCR method could specifically amplify the Chikungunya virus nucleic acid. The size of the target fragment was consistent with the expected result. The minimum detectable amount of nucleic acid was 56.7 pg. The result of Blast comparison was consistent with the result of CHIKV JC2012 sequence published by Genebank. At the same time, the CODEHOP primer binding sites were found in all the 22 alphaviruses obtained from GenBank, and the amplified products ranged in size from 510 bp to 550 bp. Conclusion The established method of RT-PCR detection of alphavirus CODEHOP is sensitive and specific and can be used for the detection of alphavirus.