应用通用引物聚合酶链反应技术检测不同型别的登革病毒

Detection or Dengue Viruses or Different Serotypes by Using Universal Primers in RT-PCR

利用特异抗体吸附的登革病毒，经含ＴｒｉｔｏｎＸ－１００的裂解缓冲液处理，释放出基因组ＲＮＡ，在同一管中进行逆转录聚合酶链扩增反应。采用这种方法利用保守性引物可检测出登革１型、２型和４型病毒参考株及我国海南岛登革２型病毒分离株的基因组序列，扩增产物约为５５０ｂｐ。用３２Ｐ－标记的分子克隆的登革２型病毒基因探针进行Ｓｏｕｔｈｅｒｎ印迹杂交分析证实了扩增产物的特异性。利用这种技术还可从感染后２４ｈ的Ｃ６／３６细胞培养上清中检测出登革病毒基因组序列。而明显的细胞病变作用（ＣＰＥ）在感染后９６ｈ才可观察到。

Dengue virions associated by their specific antibodies were treated with a buffer solution containing Triton X-100 to release viral RNAs, and then a reverse transcriptase-polymerase chain reaction (RT-PCR) of viral RNA was carried out in a single reaction vessel, By using this method, the sequences of classical of dengue type, 1,2,and 4 virused and dengue type 2 virus isolated from human sear in Hainan Island were detected by using consensus primers for NS1 genes of all 4 dengue serotypes in the RTPCR. The amplified product was about 550bp in length. the specificity was verified by performing the Southern blot hybridization of the ammplified product ot 32P-labeled cDNA probes specific for the dengue type 2 virus. The sequence of dengue-2 viral RNA could also be detected from the infected culture fluid of C6/36 cells 24hrs after virus inocalation. The clear cytopathic effect (CPF) could be observed with naked eye 96hrs after inoculation.