EvaGreen多重荧光RT-PCR结合熔解曲线分析同时检测3种呼吸道病毒  

Eva Green-based multiplex fluorescence RT-PCR with melting curve analysis for simultaneous detection of three respiratory viruses

作　　者：王琪 黄翩翩[2] 贾晓芸[2] 徐金花 方海荣 周小坚[3] 苏影[3] 莫秋华[3] WANG Qi;HUANG Pian-pian;JIA Xiao-yun;XU Jin-hua;FANG Hai-rong;ZHOU Xiao-jian;SU Ying;MO Qiu-hua(Zhuhai SHENGMA medical examination co.LTD.,Zhuhai,Guangdong 519020,China)

机构地区：[1]珠海生码医学检验有限公司,广东珠海519000 [2]珠海市妇幼保健院 [3]珠海国际旅行卫生保健中心

出　　处：《中国国境卫生检疫杂志》2018年第4期229-232,共4页Chinese Journal of Frontier Health and Quarantine

基　　金：广东省医学科学技术研究基金项目(A2017443)

摘　　要：目的建立能同时检测甲型流感病毒(Flu-A)、乙型流感病毒(Flu-B)和鼻病毒(HRV)的Eva Green多重荧光RT-RCR熔解曲线分析方法。方法针对Flu-A的M基因、Flu-B的NS1基因、HRV的5’NCR设计检测引物,并使3个基因扩增产物的解链温度(T_m值)相差≥1.5℃。采用Eva Green新型荧光染料,通过优化反应条件建立一步法多重RT-PCR反应体系。通过与商品化的荧光RT-PCR试剂盒的对照试验,对新建方法进行性能验证。结果 3种病毒的PCR产物均获得特异性的熔解曲线峰,T_m值范围分别为:Flu-A,85.0~85.5℃;Flu-B,82.5~83.0℃;HRV,88.5~89.5℃,可以通过T_m值差异明显区分3种病毒。100份临床样本的性能验证显示,新方法对3种病毒的检测均有较高的灵敏度、特异性和检验效能。结论针对3种常见呼吸道病毒建立了基于Eva Green结合熔解曲线分析的多重荧光RT-PCR方法。该方法简便实用、成本低廉,为喘息性疾病的临床诊断和病原谱研究奠定了方法学基础。Objective To establish an Eva Green-based multiplex fluorescence RT-PCR with melting curve analysis for simultaneous detection of three respiratory viruses. Methods The M gene of influenza A virus,NS1 gene of influenza B virus and 5＇ NCR of human rhinovirus（HRV） were selected to design the detection primers,and the difference between each melting temperatures（Tm） of three gene amplification products must be made into more than 1.5℃. Using a novel fluorescent dye of Eva Green, one-step multiplex RT-PCR reaction system was established by optimizing reaction conditions. The performance verification test for the new method was carried out by comparing with the commercial fluorescence RT-PCR kit. Results All of the PCR products of three viruses obtained comparatively specific peaks of melting curve at a Tm in the range of 85.0-85.5 ℃ for Flu-A,82.5-83.0℃ for Flu-B and88.5-89.5℃ for HRV,respectively. Three viruses could be clearly distinguished by the different Tmvalue. The test results of 100 clinical samples showed that the new method had high sensitivity,specificity and test effectiveness for detection of these 3 viruses. Conclusion It was the first Eva Green-based multiplex fluorescence RT-PCR assay with melting curve analysis developed for simultaneous detection of three common respiratory viruses. It was showed that this assay was a simple,reliable and cost-effective method that could be used for clinical diagnosis and pathogenic spectrum research for wheezing diseases.

关 键 词：EvaGreen染料 多重荧光RT-PCR 熔解曲线 呼吸道病毒 

分 类 号：R373.1[医药卫生—病原生物学]