应用含内参的多重实时荧光RT-PCR方法快速检测登革病毒和基孔肯雅病毒

Rapid detection of Dengue virus and Chikungunya virus by multiplex real-time RT-PCR assay with an internal control

目的建立一种登革病毒、基孔肯雅病毒并含人类基因内参检测的多重实时荧光RT-PCR方法,能在同一反应管内同时检测目前发现的所有来源的登革病毒或基孔肯雅病毒。方法针对登革病毒3′端非编码区和基孔肯雅病毒结构蛋白E2-6K-E1区以及人体各类组织细胞中均能稳定表达的RNAse P基因,设计了3套特异性引物和探针,建立了1套能同时检测登革病毒、基孔肯雅病毒及含有人类基因检测内参的多重实时荧光RT-PCR方法,对其灵敏性和特异性进行了验证,并对临床发热病人标本进行了应用评估。结果该方法对检测体外转录合成的登革病毒和基孔肯雅病毒RNA的灵敏性可达最低每个反应10～100拷贝,对检测登革1型病毒和基孔肯雅病毒的灵敏性分别可达最低每个反应0.1TCID50/mL和1TCID50/mL。用20株登革病毒、4株基孔肯雅病毒和日本脑炎病毒、西尼罗病毒、黄热病毒、盖塔病毒、辛德毕斯病毒各1株进行检测,方法的特异性均为100%。方法应用于189份发热病人血清标本检测,可准确地鉴定出其中登革病毒或基孔肯雅病毒核酸阳性的标本,且所有血清标本均能被内参引物和探针有效地扩增和杂交。结论本研究建立了一种高灵敏性、高特异性且含人类基因内参检测的登革病毒和基孔肯雅病毒多重实时荧光RT-PCR检测方法,可作为登革热或基孔肯雅热病人早期快速鉴别诊断的有效工具,也可用于蚊媒携带登革病毒或基孔肯雅病毒的高通量快速筛查。

The purpose was to establish a multiplex real-time RT-PCR assay for detection of Dengue virus and Chikun- gunya virus in one tube, which could detect all Dengue virus or Chikungunya virus strains from different origins. Based on se- quences of 3＇-UTR of Dengue virus, E2-6K-E1 region of Chikungunya virus＇s structural protein and RNAse P gene which stab- ly expressed in all human organs, 3 pairs of primers and probes were designed and a multiplex real-time RT-PCR assay to detect Dengue virus and Chikungunya virus together with human gene as reference in one tube was established. Then the specificity and sensitivity were verified, and by using the serum samples collected from fever patients, an applied evaluation of the assay was performed. Results demonstrated that the multiplex real-time RT-PCR assay could detect 10 to 100 copies per reaction for Dengue viral and Chikungunya viral in vitro transcribed RNA. For detection of Dengue virus type I and Chikungunya virus, the sensitivity could reach to minimum of 0. 1 TCIDs0/mL and 1 TCID50/mL, respectively. A specificity of 100% could be ob- served, with this assay testing on 20 Dengue virus strains, 4 Chikungunya virus strains, 1 strain of Japanese encephalitis virus, 1 strain of West Nile virus, 1 strain of yellow fever virus, 1 strain of Getah virus, and 1 strain of Sindbis virus. Of 189 serum samples from patients with fever were tested by the assay, Dengue virus or Chikungunya virus nucleic acid positive samples could be accurately identified. Furthermore, all serum samples were efficiently amplified by reference primers and hybridizedwith probes. It＇s suggested that this study established a multiplex real-time RT-PCR assay with high sensitivity, high specificity and using human gene as internal control for detecting Dengue virus and Chikungunya virus. This assay will become an effective toolfor rapid differential diagnosis of early stage of Dengue fever or Chikungunya fever. It also could be used for high throughput and rapid sereening of mosquito-carried Dengue virus or Chikungunya virus.