荧光定量RT-PCR快速检测人副流感病毒3型的研究

STUDY ON THE RAPID DETECTION OF HUMAN PARAINFLUENZA VIRUS-3 BY REAL-TIME QUANTITATIVE RT-PCR

[目的]利用荧光定量RT-PCR技术建立一种快速检测人副流感病毒3型的方法。[方法]根据人副流感病毒3型NP基因的相对保守序列分别设计一对引物及Taqman探针,建立、优化反应体系后,利用10倍稀释法检验方法的灵敏度并建立定量标准曲线;特异性检验后利用临床标本与传统的细胞培养法进行比较。[结果]人副流感病毒3型检测反应的灵敏度为0.016TCID50,标准曲线相关系数为1.00,扩增效率为89.8%,6种非人副流感病毒3型病原体检测均为阴性,说明此方法具有很高的准确性、稳定性和特异性。[结论]本研究建立荧光定量RT-PCR技术可以准确检测人副流感病毒3型,不仅灵敏度高、稳定性好,而且可以对病毒滴度进行定量检测。

[Objective] To establish a rapid method for detection of human parainfluenza virus-3 by real-time quantitative RT-PCR.Methods According to the related conserved sequence of the NP gene of human parainfluenza virus-3,a pair of primers and Taqman probe were designed and established.After optimizing the reaction system,the sensitivity of detection method were detected by tenfold serial dilution of TCID50 as well as the quantitation standard curve were established;After detection of specificity,the clinical specimen and traditional cell culture were compared.Results The sensitivity of the detection for human parainfluenza virus-3 was 0.016 TCID50,correlation coefficient of standard curve was 1.00,and the efficiency of amplification was 89.8% as well as the detection of pathogen for six kinds of human parainfluenza virus-3 showed to be negative,which demonstrated that this method with high sensibility precise,stability and specificity.Conclusion The detection system basing on real-time RT-PCR with the aspects of high sensibility and stability could precisely detect the human parainfluenza virus-3,which could be used to quantitative detection of virus titer.