人副流感病毒HPIV多重RT-PCR快速检测方法的建立

Construction of rapid multiplex reverse transcription-PCR to detect human parainfluenza viruses 1, 2, 3, and 4

目的建立快速检测人副流感病毒的多重PCR方法。方法通过查阅文献获取副流感病毒的PCR引物序列,并利用分子生物学软件对其进行评估,同时设计合成半巢式PCR引物。提取分离病毒的总RNA,以RNA抽提物为模板,一步法一次反转录扩增HPIV-1,2,3,4等4种型别的副流感病毒。以多重RT-PCR产物作为模板,用半巢式PCR进一步扩增确认。结果利用多重RT-PCR方法一次扩增出人副流感病毒的4个不同型别,条带大小分别是317、507、189和451bp,与目的片段大小一致;半巢式PCR扩增出相应的特异条带,条带大小分别是261、340、145和390bp,扩增结果与目的片段大小一致。结论人副流感病毒多重RT-PCR快速检测方法已初步建立。

Objective To construct rapid multiplex RT-PCR detection method of human parainfluenza viruses. Methods Multiplex RT-PCR primes of human parainfluenza viruses were obtained based on literature review and evaluated by molecular biologic software, then designed and synthesized half-nested PCR primers. Total viral RNA was extracted as templates of multiplex RT-PCR and then reverse transcribed to expand HPIV-1 ,HPIV-2, HPIV-3 and HPIV-4 respectively in one tube by OneStep RT-PCR. The multiplex RT-PCR products were further confirmed by half-nested PCR. Results The sizes of expansion bands of 4 different types of HPIVs were 317,507,189 and 451bp respectively by multiplex RT-PCR, and consistent with target segments. The multiplex RT-PCR products amplified by half-nested PCR were 261,340,145 and 390bp respectively and also had consistency. Conclusion Rapid multiplex RT-PCR method was successfully constructed to detect human parainfluenza viruses type 1,2,3 and 4.