Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan 45320.
Poult Sci. 2009 Dec;88(12):2526-31. doi: 10.3382/ps.2009-00262.
A multiplex reverse transcription-PCR (mRT-PCR) was developed and standardized for the detection of type A influenza viruses, avian influenza virus (AIV) subtype H7, H9, and H5 hemagglutinin gene with simultaneous detection of 3 other poultry respiratory pathogens, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV). Seven sets of specific oligonucleotide primers were used in this study for the M gene of AIV and hemagglutinin gene of subtypes H7, H9, and H5 of AIV. Three sets of other specific oligonucleotide primers were used for the detection of avian respiratory pathogens other than AIV. The mRT-PCR DNA products were visualized by agarose gel electrophoresis and consisted of DNA fragments of 1,023 bp for M gene of AIV, 149 bp for IBV, 320 bp for NDV, and 647 bp for ILTV. The second set of primers used for m-RT-PCR of H7N3, H9N2, and H5N1 provided DNA products of 300 bp for H7, 456 bp for H5, and 808 bp for H9. The mRT-PCR products for the third format consisted of DNA fragments of 149 bp for IBV, 320 bp for NDV, 647 bp for ILTV, 300 bp for H7, 456 bp for H5, and 808 bp for H9. The sensitivity and specificity of mRT-PCR was determined and the test was found to be sensitive and specific for the detection of AIV and other poultry respiratory pathogens. In this present study, multiplex PCR technique has been developed to simultaneously detect and differentiate the 3 most important subtypes of AIV along with the 3 most common avian respiratory pathogens prevalent in poultry in Pakistan. Therefore, a mRT-PCR that can rapidly differentiate between these pathogens will be very important for the control of disease transmission in poultry and in humans, along with the identification of 3 of the most common respiratory pathogens often seen as mixed infections in poultry, and hence economic losses will be reduced in poultry.
建立并标准化了一种多重逆转录聚合酶链反应（mRT-PCR），用于检测 A 型流感病毒、禽流感病毒（AIV）亚型 H7、H9 和 H5 血凝素基因，并同时检测其他 3 种家禽呼吸道病原体，即新城疫病毒（NDV）、传染性支气管炎病毒（IBV）和传染性喉气管炎病毒（ILTV）。本研究使用了 7 组特异性寡核苷酸引物，用于 AIV 的 M 基因和 AIV 亚型 H7、H9 和 H5 的血凝素基因。另外 3 组特异性寡核苷酸引物用于检测除 AIV 以外的其他家禽呼吸道病原体。mRT-PCR 的 DNA 产物通过琼脂糖凝胶电泳可视化，包括 AIV 的 M 基因的 1023bp、IBV 的 149bp、NDV 的 320bp 和 ILTV 的 647bp 的 DNA 片段。用于 H7N3、H9N2 和 H5N1 的 m-RT-PCR 的第二组引物提供了 300bp 的 H7、456bp 的 H5 和 808bp 的 H9 的 DNA 产物。第三组 mRT-PCR 产物由 149bp 的 IBV、320bp 的 NDV、647bp 的 ILTV、149bp 的 IBV、320bp 的 NDV、647bp 的 ILTV、300bp 的 H7、456bp 的 H5 和 808bp 的 H9 的 DNA 片段组成。确定了 mRT-PCR 的敏感性和特异性，发现该试验对检测 AIV 和其他家禽呼吸道病原体具有敏感性和特异性。在本研究中，开发了多重 PCR 技术，用于同时检测和区分巴基斯坦家禽中最常见的 3 种 AIV 亚型以及最常见的 3 种家禽呼吸道病原体。因此，一种能够快速区分这些病原体的 mRT-PCR 将对控制家禽和人类中的疾病传播非常重要，同时还能识别家禽中常见的混合感染的 3 种最常见的呼吸道病原体，从而减少家禽的经济损失。
