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一种黄热病毒反转录恒温热隔绝式PCR检测方法

A reverse transcription-insulated isothermal PCR method for the detection of Yellow fever virus
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摘要 目的建立能在野外或现场应用的黄热病毒反转录恒温热隔绝式PCR(reverse transcription-insulated isothermal PCR,RT-iiPCR)快速检测方法。方法针对黄热病毒基因组5’UTR保守区设计检测引物和TaqMan-MGB探针,优化引物、探针、Taq酶、反转录酶和PCR增强剂等各组分的浓度,利用分析平台,建立黄热病毒RT-iiPCR检测方法,评价方法的稳定性、特异性、灵敏度和检测限等指标。结果建立了稳定可靠的黄热病毒RT-iiPCR检测方法,在分析平台上只需一键操作即可在58 min内完成检测并自动报告结果。性能评价试验显示该方法稳定性好,特异性和灵敏度分别达到100.00%和92.31%,检测下限≥1 000拷贝。结论率先建立了黄热病毒RT-iiPCR检测方法,该方法简便实用,适合于现场快速检测。 Objective To develop a reverse transcription-insulated isothermal PCR(RT-iiPCR)method for rapid and on-site detection of Yellow fever virus(YFV).Methods The 5’UTR conserved region of YFV genome was selected for primers and TaqMan-MGB probe design.The optimized reaction conditions,such as concentration of primers,probe,Taq DNA polymerase,reverse transcriptase and PCR enhancer reagents were conducted.The RT-iiPCR assay was established on the POCKITTManalysis platform.The performance parameters such as stability,specificity,sensitivity and the detection limit were evaluated.Results A stable and reliable RT-iiPCR method for the detection of YFV was obtained.The detection could be completed within 58 minutes with one-click operation on the POCKITTM analysis platform,the positive and negative results were automatically reported on the display screen.The perfor-mance evaluation showed that the method was stable,with the specificity and sensitivity up to 100.00%and 92.31%,respectively,and the lowest detection limit was≥1000 copies of target RNA template.Conclusion This is the first development of RT-iiPCR method for the detection of YFV.The method is simple,practical and suitable for rapid on-site detection.
作者 赵俊华 李伟刚 杜田 苏影 汪海波 涂承宁 杨泽 莫秋华 ZHAO Jun-hua;LI Wei-gang;DU Tian;SU Ying;WANG Hai-bo;TU Cheng-ning;YANG Ze;MO Qiu-hua(Zhuhai International Travel Healthcare Center,Zhuhai,Guangdong 519020,China;不详)
出处 《中国国境卫生检疫杂志》 CAS 2019年第4期229-232,共4页 Chinese Journal of Frontier Health and Quarantine
基金 海关总署科研计划项目(2018IK045,2017IK212) 广东省科技计划项目(2018B020207013).
关键词 恒温热隔绝式PCR 黄热病毒 黄病毒 现场检测 Insulated isothermal PCR Yellow fever virus Flavivirus On-site detection
作者简介 通信作者:莫秋华,E-mail:mqhzh13@163.com
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