酶促重组等温扩增实时荧光法快速检测基孔肯雅病毒方法的建立

Development of real-time fluorescent enzymatic recombinase amplification method for chikungunya virus detection

目的建立一种快速、准确、高灵敏度检测基孔肯雅病毒(chikungunya virus,CHIKV)的逆转录-酶促重组等温扩增(reverse transcription enzymatic recombinase amplification,RT-ERA)的基因检测技术方法.方法以CHIKV非结构蛋白1(non-structural protein 1,NSP1)基因的保守序列为靶标,根据RT-ERA反应原理设计、合成荧光探针及对应引物,以pUC18为载体构建靶标质粒并在体外转录后作为阳性对照品,将阳性对照品倍比稀释后筛选出扩增效率最高的探针引物组合.利用筛选的探针引物组合,对不同拷贝数的CHIKV进行荧光RT-ERA法扩增,评价该方法的最低检出限;再分别以森林脑炎病毒、登革热病毒、寨卡病毒、乙型脑炎病毒、新布尼亚病毒的RNA为模板进行荧光RT-ERA法扩增,评价该方法的特异性.结果在40℃恒温条件下,利用荧光RT-ERA法10 min内可以完成CHIKV核酸扩增,该方法最低检出限可达10拷贝/μL,且当CHIKV核酸扩增为阳性时,其他病毒检测结果为阴性.结论成功建立一种可用于检测CHIKV NSP1基因的荧光RT-ERA法,该方法操作简便、检测限低且特异性好.

Objective To establish a rapid,accurate and highly sensitive detection method for chikungunya virus based on reverse transcription-enzyme recombinase amplification(RT-ERA)method.Methods Using the conserved sequence of chikungunya virus non-structural protein 1(NSP1)gene as the target,the fluorescent probes and primers were designed and synthesized according to the RT-ERA principle.The target plasmid was constructed using pUC18 as the carrier and transcribed in vitro.The product was used as the positive control.The combination of primers and fluorescent probes with the highest amplification efficiency was screened using serially diluted positive control.The combination of selected probe and primers was used in the fluorescent RT-ERA method to amplify chikungunya virus of different copy numbers to assess the lower detection limit of the method.The RNA templates of forest encephalitis virus,dengue virus,West Nile virus,Japanese encephalitis virus and yellopw fever virus were amplified by fluorescent RT-ERA to assess the specificity of the method.Results The fluorescent RT-ERA method could effectively amplify chikungunya virus RNA within 10 min at 40℃and the lower detection limit was 10 copies/μL.The detection results of other viruses and only chikungunya virus was positive.Conclusions A fluorescence RT-ERA method for detection of Chikungunya virus NSP1 gene has been successfully established.The method is simple with low detection limit and good specificity.