LNA探针同时检测人副流感病毒1，2，3型多重荧光定量RT—PCR方法的建立

Simultaneous detection of human parainfluenza viruses 1, 2, 3 by multiplex real-time reverse transcription-polymerase chain reaction with LNA probes

目的人副流感病毒1，2，3型是呼吸道感染的主要病原。本研究建立了特异、快速、灵敏的多重荧光定量RT—PCR方法用于人副流感1，2，3型病毒临床标本检测。方法针对人副流感1，2，3型病毒设计特异性引物探针，优化荧光RT—PCR反应条件。应用体外转录方法分别制备人副流感1，2，3型病毒的标准品。验证荧光定量RT-PCR方法的特异性，敏感性和稳定性。结果该方法对人副流感1，2，3型病毒核酸检测有高度特异性，检测的灵敏度HPIV1为10个拷贝，HPIV2为100个拷贝，HPIV3为100个拷贝。可从临床患者鼻咽吸出物标本中直接检出。结论’本研究建立的LNA探针同时检测人副流感病毒1，2，3型多重荧光定量RT-PCR方法具有较高的特异性和敏感性。适用于临床早期诊断和实验室病原谱筛查。

Objective Human parainfluenza virus （HPIV） types 1, 2 and 3 are major viral pathogens responsible for upper and lower respiratory tract infections. In this study, a real-time RT-PCR was developed using multiplex primers-probe （ HPIV-1,2,3 ） for the simuhaneous detection of both HPIV1, HPIV2 and HPIV3 genomes. Methods Optimal primers and probes were designed using specialized software. The conditions for multiplex real-time RT-PCR had been optimized. The synthesis of RNA standards of HPIV1,2,3 were used a T7 RNA polymerase. Check the specificity sensitivities and stability of one step RT-PCR assay. Results Obtained in a 10-fold dilution series assay demonstrate a high sensitivity of the assay with a lowest detection limit of 10 copies for HPIV1, 100 copies for HPIV2 and 100 copies for HPIV3. Conclusion The assays demonstrates an improved sensitivity and scope of detecting HPIV1,2,3 viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the pre-diagnosis and respiratory virus pathogenesis.