双重荧光定量RT-PCR快速检测N1、N2亚型流感病毒的研究

Rapid detection of the N_1 and N_2 subtype of influenza virus by duplex fluorescence quantitative RT-PCR

目的利用荧光定量RT-PCR技术建立快速检测流感病毒N1、N2亚型的方法。方法根据N1、N2亚型流感病毒NA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,采用十倍稀释体外转录RNA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 N1、N2亚型流感病毒的检测灵敏度为10拷贝/μl,扩增效率分别为102.21%和101.78%,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。结论双重荧光定量RT-PCR技术可以快速、准确的检测N1、N2亚型流感病毒。

Objective To develop a method to rapidly detect the N1 and N2 subtype of influenza virus by fluorescence real-time quantitative PCR. Methods According to the conservative sequences of NA gene of the N1 and N2 subtype of influenza virus,two pairs of primer and Taq-man probe were designed,respectively. Using one step RT-PCR kit to set up a duplex RT-PCR system for detecting the N1 and N2 subtype of influenza virus; the standard quantitative curve of the assay was established by using 10-fold serial dilution of in-vitro transcribed RNA, and the sensitivity and reproducibility was determined. The specificity and test for influenza virus and re＇spirovirus were also determined by using this duplex Real-time RT-PCR system. Results The sensitivity of detecting the N1 and N2 subtype of influenza virus was 10 eopies/μl, and the regression coefficient of the quantitative curve was higher than 99%. The amplification efficiency and specificity of this assay was 102.21% and 101.78% respectively. The system was capable of detecting human influenza viruses with high specificity. Conclusion The detection system based on RT-PCR could be utilized to rapidly and sensitively detect the N1 and N2 subtype of influenza virus.