Saberfar Esmaeil, Forghani-Fard Mohammad M, Mosavi Mirlatif
Dept. of Microbiology and Biological Centre, Faculty of Medicine, Baqiyatallah (a.s.), University of Medical Sciences, Tehran, Iran.
Dept. of Biology, Shahed University, Tehran, Iran
Iran Biomed J. 2007 Apr;11(2):69-74.
Avian influenza virus (AIV) infection is a major cause of bird or human mortality and morbidity, therefore the rapid identification of the virus is of important clinical and epidemiological implication.
A multiplex Reverse Transcriptase PCR (RT-PCR) was optimized for the detection of influenza A virus and the H5 and H9 subtypes. The influenza type A specific primers were directed to the region of the influenza A matrix gene that is conserved among most type A influenza viruses. The H5 and H9 primers were directed to H5 and H9 hemagglutinin (HA) gene regions that are conserved among H5 and H9 subtypes. The selected primer sets were used in the RT-PCR for simultaneous detection of matrix, H5 and H9 responding specific sequences in a multiplex format.
Three reaction conditions were optimized which include: i) RT-PCR typing using matrix gene primers for five subtypes of flu A (H1, H3, H5, H7 and H9), ii) RT-PCR subtyping for H5 and H9 subtypes, and iii) multiplex subtyping of H5 and H9. In this study, the multiplex RT-PCR was applied to 147 cloacal and tracheal swabs of clinical poultry cases with similar influenza symptoms.
These results suggest that multiplex RT-PCR assay can be a useful test for rapid detection and subtyping of AIV in clinical samples.
禽流感病毒（AIV）感染是禽类或人类死亡和发病的主要原因，因此快速鉴定该病毒具有重要的临床和流行病学意义。
优化了多重逆转录聚合酶链反应（RT-PCR）以检测甲型流感病毒以及H5和H9亚型。甲型流感特异性引物针对大多数甲型流感病毒中保守的甲型流感病毒基质基因区域。H5和H9引物针对H5和H9亚型中保守的H5和H9血凝素（HA）基因区域。所选引物组用于RT-PCR，以多重形式同时检测基质、H5和H9的相应特异性序列。
优化了三种反应条件，包括：i）使用基质基因引物对甲型流感的五个亚型（H1、H3、H5、H7和H9）进行RT-PCR分型，ii）对H5和H9亚型进行RT-PCR亚型分型，以及iii）对H5和H9进行多重亚型分型。在本研究中，多重RT-PCR应用于147份具有相似流感症状的临床家禽病例的泄殖腔和气管拭子。
这些结果表明，多重RT-PCR检测可作为临床样本中AIV快速检测和亚型分型的有用检测方法。
