实时荧光RT-PCR快速检测蚊体内乙型脑炎病毒及福建省基因Ⅰ型乙型脑炎病毒的发现  

Rapid detection of Japanese encephalitis virus from mosquitoes by real-time fluorescence RT-PCR and discovery of genotype Ⅰ Japanese encephalitis virus in Fujian Province

作　　者：郑夔[1] 黄吉城[1] 李小波[1] 洪烨[1] 师永霞[1] 幸芦琴[1] 相大鹏[1] 郭波旋[1] 胡龙飞 

机构地区：[1]广东检验检疫技术中心卫生检疫实验室,广州510700 [2]云浮出入境检验检疫局,广东云浮527300

出　　处：《中国卫生检验杂志》2008年第11期2212-2215,共4页Chinese Journal of Health Laboratory Technology

基　　金：2005年度国家质检总局科技计划项目(2005IK-079)

摘　　要：目的:应用实时荧光RT-PCR方法快速检测蚊体内乙型脑炎病毒,并对蚊体内携带的乙型脑炎病毒进行基因分型。方法:从蚊媒监测点采集蚊标本,研磨后提取RNA,用新型的LNA探针实时荧光RT-PCR方法进行高通量快速筛查蚊体携带的乙型脑炎病毒,对核酸阳性的标本进一步用乙型脑炎病毒E基因特异引物进行RT-PCR扩增和序列测定,根据所测序列与GenBank中不同地理来源乙型脑炎病毒株E基因核苷酸序列分析的结果进行基因型别的判定。结果:12575只蚊子分成254份,用实时荧光RT-PCR方法从来源于福州的2份三带喙库蚊标本中检出乙型脑炎病毒核酸阳性,经RT-PCR及核苷酸序列测定,得到一段长1500nt的E基因核苷酸序列,序列分析结果显示,其核苷酸同源性最大的是来源于上海(DQ404100)和浙江(EU258742)的毒株,分别是99.1%和99.0%,均属于基因Ⅰ型病毒。结论:实时荧光RT-PCR方法是快速检测蚊体内乙型脑炎病毒的理想方法,此次调查发现的乙型脑炎病毒是以往福建省内未见报道的基因Ⅰ型病毒。Objective： To rapidly detect Japanese encephalitis virus from mosquitoes by real - time fluorescence RT - PCR assay. And to analyse the genotype of Japanese encephalitis virus carried from mosquitoes. Methods： Mosquitoes were collected from monitoring points. RNA was extracted after grinding. A novel LNA probe real - time fluorescence RT - PCR assay was used for a high - throughput and rapid screening of Japanese encephalitis virus carried from mosquitoes. Then the nucleonic acid detection of positive samples was performed with RT - PCR amplification and sequenced by using primers specific to E gene of Japanese encephalitis virus. The E gene sequence obtained was compared with the sequences of E gene of various geography source strains obtained in GenBank, according to the result of nucleotide sequence analysis. The genotype of Japanese encephalitis virus was determined. Results：A total of 12575 mosquitoes were collected and were divided into 254 groups. The real -time fluorescence RT- PCR assay showed that 2 samples of Culex tritaeniorhynchus sourced from Fuzhou City with positive results of Japanese encephalitis virus nucleonic acid detection. After RT - PCR amplification and sequencing, a length of 1500nt sequence of E gene was gotten. Aligning the sequence with the GenBank database, a maximum nucleotide homology with Shanghai strain （DQ404100） and Zhejiang strain （EU258742） could be seen, the identity of which was 99. 1% and 99.0%, respectively. All of the strains belong to genotype Ⅰ . Conclusion： Real - time fluorescence RT - PCR assay is an ideal method for rapid detection of Japanese encephalitis virus from mosquitoes. The discoverable Japanese encephalitis virus in this investigation belongs to genotype Ⅰ , which has never been reported in Fujian Province.

关 键 词：实时荧光RT—PCR LNA探针 乙型脑炎病毒 E基因 基因型 

分 类 号：R373.31[医药卫生—病原生物学]