双重荧光定量RT-PCR快速检测流感病毒H1、H3亚型的研究

Rapid detection of influenza virus H1 and H3 subtype by duplex fluorescence quantitative RT-PCR

目的利用荧光定量RT-PCR技术建立一种快速检测流感病毒H1、H3亚型的方法。方法根据H1、H3亚型流感病毒HA基因的相对保守序列,设计两对引物及其相应的Taqman探针,利用一步法RT-PCR试剂盒建立优化反应体系后,将荧光定量RT-PCR的产物采用10倍稀释法,即107～100copies/μl,再次用荧光定量PCR方法检验建立体系的灵敏度和重复性,并建立相对定量标准曲线;利用多种流感病毒和具有相似临床症状的呼吸道病毒检验建立体系的特异性。结果 H1和H3亚型流感病毒的检测灵敏度为102copies/μl,扩增效率分别为101.35%和113.28%,标准曲线相关系数大于99%,重复性良好,特异度实验未发现有非特异性扩增。结论本研究建立的双重荧光定量RT-PCR技术可以快速、准确地检测H1、H3亚型流感病毒。

Objective To develop a method to rapidly detect influenza virus H1 and H3 subtype by fluorescence real-time quantitative RT-PCR. Methods According to conservative sequences of HA gene of influenza virus H1 and H3 subtype, two sets of primers and Taq-man probes were designed. One step RT-PCR kit was used to set up duplex real-time RT-PCR system for detection of influenza virus H1 and H3 subtype.The standard quantitative curve of the assay was established.10-fold serial dilution of in-vitro transcribed RNA was used in the detection of the sensitivity and reproducibility of this system.The specificity of this system was also determined by testing for other influenza virus and respirovirus. Results The sensitivity of detection influenza virus H1 and H3 subtype was 102 copies/μl and the regression coefficient of the quantitative curve exceeded 99%.The amplification efficiency and specificity of this assay was 101.35% and 113.28%,respectively.The system was capable of detecting human influenza viruses with high specificity. Conclusion The detection system based on real-time RT-PCR could be utilized to rapidly and sensitively detect influenza virus H1 and H3 subtype.