摘要
目的：检测血清中的登革热（DF）病毒．并进行分型研究。方法：采用SiO2快速提取DF病毒RNA，用通用引物（D1～D4型）RT－PCR扩增方法，检测广东省1995年55份疑似DF患者急性期（1～7天）血清标本中的DF病毒RNA，并与病毒分离法相比较。结果；病毒分离阳性率为49．09％，RT－PCR的检出率为38．18％，敏感性为77．78％，特异性为100％，符合率为89．09％，两者差异无显著性（X2＝2．08，P＞0．05）。用通用引物扩增D1～D4型标准毒株和标本，出现511bp的扩增带，乙脑病毒不出现扩增带。用HinfⅠ、HaeⅢ、HincⅡ和Sau3aⅠ等4种限制性内切酶，对D1～D4型标准毒株的RT－PCR产物进行酶切分型，用琼脂糖电泳，结果HinfⅠ和HaeⅢ对D1～D4型均能酶切，Sau3aⅠ能酶切D1～D3型，HincⅡ能酶切D1和D4型。根据两种或以上的酶切结果，可以对DF病毒的型别作出鉴定。用这4种酶对5份标本进行酶切分型，结果与D1型标准毒株的电泳带型相同。结论：用本方法检出DF病毒的RNA仅需5～6小时，在2天内可以鉴定出型别，是诊断DF病毒感染的一种快速简单的方法。
Dengue viruses RNA was extracted rapidly by using silica. Dengue virus consensusprimers of types 1-4 and reverse transcriptase-polymerase chain reaction (RT-PCR) were usedfor detecting dengue virus in 55 serum specimens collected from patients with suspected denguefever in acute phase in Guangdong Province in 1995,and compared with the method of virus isolation. The results showed that the dengue viruses RNA detecting positive rate by RT-PCR was 38.18%, the sensitivity was 77. 7 %, the specificity was 100%, and the agreement rate was 89. 09%.The dengue viruses positive rate by using virus isolation was 49. 09%,without significant differencecompared with the one in RT-PCR (X2=2.08,P>0. 05). The amplified bands of 511bp were observed in amplified products of the reference viruses strains of types 1, 2, 3,and 4,and specimensrespectively,by consensus primers,but not observed in Japanese encephalitis virus. The amplifiedproducts of dengue viruses by RT-PCR were cleaved by restriction endonucleases of Hinf Ⅰ,HaeⅢ,Hinc Ⅱ and San 3a Ⅰ,respectively,on agarose gel electrophoresis for differentiation among fourtypes of dengue viruses. The results showed that amplified products of dengue viruses of types Ⅰ- 4 were cut by Hinf Ⅰ and Hae Ⅲ, the products of types land2 were cut by Sau 3a Ⅰ, andthe products of types 1,2,and 3 were cleaved by Hinc Ⅱ. The dengue viruses of 4 types can be differentiated by the cleavage of two or more restriction endonucleases. The result of typing of fivespecimens was identical with the one of the reference dengue virus of type 1. The method of this paper takes only 5 to 6 hours to detect dengue viruses RNA,and two days to differentiate the fourtypes of dengue viruses. It is a rapid and simple method for detecting and typing dengue viruses.
出处
《中国媒介生物学及控制杂志》
CAS
CSCD
1997年第6期447-451,共5页
Chinese Journal of Vector Biology and Control
关键词
登革病毒
分型
微生物学检验
聚合酶链反应
Dengue virus
Consensus primers
Silica reverse transcriptase-polymerase chain reaction(RT-PCR)
Typing by restriction endonuclease cleaving
