中国人兽共患病学报2013，Vol.29Issue(3)：242-247,6.DOI:10.3969/cjz.j.issn.1002-2694.2013.03.007
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
摘要
Abstract
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 sequences of 3 -UTR of Dengue virus , E2-6K-E1 region of Chikungunya virus's structural protein and RNAse P gene which stably 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 TCID50 /mL and 1 TCID50 /mL, respectively . A specificity of 100% could be observed , 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 hybridized with 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 tool for rapid differential diagnosis of early stage of Dengue fever or Chikungunya fever. It also could be used for high throughput and rapid screening of mosquito-carried Dengue virus or Chikungunya virus .关键词
登革病毒/基孔肯雅病毒/内参/多重实时荧光RT-PCRKey words
Dengue virus / Chikungunya virus / internal control/ multiplex real-time RT-PCR分类
医药卫生引用本文复制引用
郑夔,丁国允,周惠琼,谢雪妹,李小波,师永霞,苏锦坤,黄吉城..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[J].中国人兽共患病学报,2013,29(3):242-247,6.基金项目
质检公益性行业科研专项基金资助项目(No.201010036) （No.201010036）
