多重逆转录-聚合酶链反应快速检测登革病毒及其临床应用

任瑞文; 方美玉; 刘建伟; 王军军; 郝丽; 程刚锋; 洪文艳; 田小东

Abstract

任瑞文,方美玉,刘建伟,王军军,郝丽,程刚锋,洪文艳,田小东.多重逆转录-聚合酶链反应快速检测登革病毒及其临床应用[J].Chinese journal of Epidemiology,2005,26(1):29-32

多重逆转录-聚合酶链反应快速检测登革病毒及其临床应用

Development of multiplex reverse translation??polymerase chain reaction methods f or detection of dengue virus type 1??4 and its application in clinical use ??

Received:January 10, 2004

DOI：

KeyWord: 登革病毒多重聚合酶链反应快速检测

English Key Word: Deng ue v irus Multiplex reverse tr anslation..polymerase chain react ion Quick detection

FundProject:全军医学科研..十五..计划重大课题资助项目(01Z014);广东省自然科学基金资助项目(04001618)

Author Name	Affiliation
REN Rui-wen	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
FANG Mei-yu	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
LIU Jian-wei	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
WANG Jun-jun	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
HAO Li	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
CHENG Gang-feng	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
HONG Wen-yan	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China
TIAN Xiao-dong	The Military Medical Institute of Guangzhou Military District, Guangzhou 510507, China

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Abstract:

目的建立登革1～4型病毒的多重逆转录聚合酶链反应(RTPCR)快速检测及分型方法。方法参照登革1～4型病毒核酸序列设计多重RTPCR引物,并检索国际基因序列数据库初步验证其特异性,随后对PCR反应条件进行优化,以同属于黄病毒科的黄热病毒、流行性乙型脑炎病毒为对照,验证其特异性。并对2003年30份临床疑似登革患者血清标本进行了检测,阳性片段克隆测序验证扩增片段特异性。结果采用多重PCR引物对登革1～4型病毒进行扩增,分别获得295、237、118、347bp片段,与设计相符;而黄热病毒及流行性乙型脑炎病毒均无非特异性扩增条带,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果,30份疑似患者血标本RTPCR扩增阳性率为83.3%(25/30),其核酸序列与登革1型病毒柬埔寨株以及中国1997、1999年流行株GD14/97、GD05/99同源性分别为97%、97%、98%。结论实验证明,所建立的多重PCR方法能够快速地检测和鉴定登革1～4型病毒,为登革病毒的检测及分型提供了一种方便易行的方法。

English Abstract:

Objective?? To develop multiplex r everse translat ion??polymerase chain r eaction( RT??PCR) method for detection of dengue vir us type 1??4. Methods?? Based on the genomes sequence analysis of dengue v irus type 1??4, four??pair o f primers were desig ned. The specificity of the primers w as pr imar ily tested by searching the GenBank DNA sequence database. The optimal r eaction conditions of the mult iplex RT??PCR w ere then established. The specificity of RT??PCR was tested using the homologous y ellow fever v irus and Japanese encephalitis virus. 30 serum samples o f dengue v irus from suspected sufferers in the prevalence of dengue virus in 2003 w er e detected using the methods we developed. Results ?? Posit ive segments about 295, 237, 118, 347 bp could be seen in the multiplex RT??PCR productio n of dengue virus ty pe 1??4, r espectiv ely. T here were no positiv e segments in the RT??PCR product ions of Japanese encephalitis virus and yellow fever v irus. 25 of the 30 serum samples showed dengue virus type 1 positive results, while the sequencing results sug gesting the amplification sequence having a high homology with dengue virus ty pe 1 strain Cambodia, GD14/ 97 and GD05/ 99( 97%, 97%, 98%, r espective ) . Conclusion?? The method of multiplex RT??PCR we established could be used for early detection and identification of dengue virus type 1??4.

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