Chungue E, Roche C, Lefevre M F, Barbazan P, Chanteau S
Institut Territorial de Recherches Médicales Louis Malardé, Papeete, Tahiti.
J Med Virol. 1993 Jun;40(2):142-5. doi: 10.1002/jmv.1890400211.
A rapid, simple and efficient single-tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 microliters) followed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Recovery of RNA is based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate in the presence of silica. The silica RT-PCR can be completed within 5 hours starting from RNA extraction to agarose gel electrophoresis. All of the 63 dengue-3 culture-positive sera were RT-PCR-positive (virus titres: < 10(2) to 11(10.69.). Of 33 culture-negative acute sera from serologically confirmed dengue fever patients collected during dengue-3 epidemic, 4 were RT-PCR-positive. RT-PCR was also positive in 29 of 30 dengue-1 culture-positive sera (virus titres range: < 10(2) to 10(8.69). Dengue-1 virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.
本文描述了一种快速、简单且高效的单管方法，用于从少量血清（10微升）中分离登革病毒RNA，随后进行逆转录聚合酶链反应（RT-PCR）。RNA的回收基于硫氰酸胍在硅胶存在下的裂解和核酸酶失活特性。从RNA提取到琼脂糖凝胶电泳，硅胶RT-PCR可在5小时内完成。63份登革3型培养阳性血清全部RT-PCR阳性（病毒滴度：<10²至10¹⁰.⁶⁹）。在登革3型流行期间收集的33份血清学确诊登革热患者的培养阴性急性血清中，4份RT-PCR阳性。30份登革1型培养阳性血清中的29份RT-PCR也呈阳性（病毒滴度范围：<10²至10⁸.⁶⁹）。通过硅胶RT-PCR在野外捕获的埃及伊蚊中也检测到了登革1型病毒。
