Figueiredo L T, Batista W C, Igarashi A
Unidade Multidisciplinar de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil.
Mem Inst Oswaldo Cruz. 1997 May-Jun;92(3):395-8. doi: 10.1590/s0074-02761997000300016.
We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50 microliters assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, and IU of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37 degrees C for RT followed by a variable amount of cycles of two-step PCR amplification (92 degrees C for 60 sec, 53 degrees C for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 10(2.8) TCID 50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.
我们在此展示一种用于鉴定登革2型病毒的简化逆转录聚合酶链反应（RT-PCR）。本研究使用了从巴西患者中分离出的三株登革2型病毒毒株，以及作为阴性对照的黄热病疫苗17DD。用病毒感染C6/36细胞，感染7天后收集组织培养液。RT-PCR是在单一反应管中单次添加试剂后将RT和PCR结合进行的，先使用1%的诺乃洗涤剂P-40对病毒进行消化。50微升的检测反应混合物包含50皮摩尔的一对登革2型特异性引物，用于扩增包膜蛋白基因的210碱基对序列、0.1毫摩尔的四种脱氧核苷三磷酸、7.5单位的逆转录酶和1单位的耐热Taq DNA聚合酶。试剂混合物在37℃孵育15分钟进行RT反应，随后进行可变数量的两步PCR扩增循环（92℃ 60秒，53℃ 60秒），温度缓慢上升。PCR产物进行1.7%琼脂糖凝胶电泳，在溴化乙锭溶液中孵育凝胶后用紫外光观察。PCR 25和30个循环后观察到DNA条带。RT-PCR可检测到低至10(2.8) TCID 50/ml的病毒量。三株登革2型毒株均观察到特异性DNA扩增。与其他RT-PCR相比，该检测方法具有以下优点：避免了繁琐的病毒RNA提取；RT和PCR的结合减少了检测时间，便于操作并降低了污染风险；两步PCR循环产生清晰的DNA扩增，节省了检测时间并简化了技术。
