用3'非编码区通用引物通过RT-PCR检测登革1～4型病毒RNA

DETECTION OF DENGUE VIRUSES TYPE 1～4 BY REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION USING 3'-NONCODING REGION UNIVERSAL PRIMERS

目的 根据登革病毒 (DEN) 3 端非编码区的一段高度保守序列 ,设计登革 1～ 4型通用引物 ,采用逆转录聚合酶链反应 (RT PCR)检测登革病毒 1～ 4型RNA。方法 用C6/ 3 6细胞培养登革病毒。用热酚法提取病毒RNA ,进行RT PCR扩增 ,并对扩增产物测序。结果 DEN 2 NGC株可扩增出至少 10TCID5 0 的病毒 ,测序结果与已知序列一致。DEN 1～ 4型标准株和DEN -2 -0 4与DEN 2 4 3株均能够扩增出唯一的一条特异条带。结论 应用通用引物RT PCR法可从病毒浓度少至 10TCID5 0 的感染细胞培养液中检出DEN 2 NGC株病毒 ,并且该对通用引物对于其他 3型登革病毒亦具有很好的通用性 ,其方法的特异性和敏感性亦较强。

Aim A pair of universal primers was designed according to a highly reserved domain of the 3 noncoding region of dengue viruses type 1～4 It was used in the RT PCR to diagnose dengue virus RNA Method Dengue viruses were cultured with C6/36 cells By using thermal phenol method to extract dengue virus RNA,those RNA samples were amplified respectively by RT PCR and the product were sequenced Result DEN 2 NGC RNA can be amplified from infected tissue culture fluid with viral concentration as low as 10TCID 50 and the amplified product accords with the DEN 2 NGCs sequence The unique target DNA bands of each products of classical dengue viruses type 1～4 and domestic dengue viruses type DEN 2 04 and DEN 2 43 also can be obtained by amplification Conclusion This pair of primers is characterized by universality and this method reflects good quality of sensitivity and specificity