Mardani Karim, Noormohammadi Amir H, Ignatovic Jagoda, Browning Glenn F
Department of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia.
Avian Pathol. 2006 Feb;35(1):63-9. doi: 10.1080/03079450500465817.
Typing infectious bronchitis virus (IBV) strains is useful for implementation of control measures and for understanding the epidemiology and evolution of IBV. The aim of the work reported here was to develop a rapid and sensitive method for typing isolates of IBV, if possible directly from tissues of infected birds. A procedure was developed for differentiation of IBV strains by restriction endonuclease fragment length polymorphism (RFLP) analysis of a 7.5 kb DNA fragment amplified from their genome by reverse transcription-polymerase chain reaction (RT-PCR). This fragment encompassed all of the genes encoding the structural proteins of the virus. Viral RNA was extracted either directly from tissues of diseased birds or from virus propagated in embryonated eggs, and was subjected to RT-PCR. Three different restriction endonucleases, AluI, Sau3AI and MnlI, were used to digest the 7.5 kb PCR product from different IBV strains and the resultant RFLP patterns were compared. Patterns obtained with all three enzymes grouped IBV strains belonging to the same serotype in the same cluster. These results show that the RT-PCR-RFLP system described here can be used as a quick and inexpensive tool for differentiating IBV strains.
对传染性支气管炎病毒（IBV）毒株进行分型，有助于实施防控措施，也有助于了解IBV的流行病学和进化情况。本文报道的这项工作的目的是开发一种快速且灵敏的方法，用于对IBV分离株进行分型，如有可能，直接从感染禽类的组织中进行分型。开发了一种程序，通过对经逆转录-聚合酶链反应（RT-PCR）从其基因组中扩增出的7.5 kb DNA片段进行限制性内切酶片段长度多态性（RFLP）分析，来区分IBV毒株。该片段包含了病毒所有编码结构蛋白的基因。病毒RNA可直接从患病禽类的组织中提取，也可从在鸡胚中增殖的病毒中提取，然后进行RT-PCR。使用三种不同的限制性内切酶AluI、Sau3AI和MnlI，对来自不同IBV毒株的7.5 kb PCR产物进行酶切，并比较所得的RFLP图谱。用这三种酶获得的图谱将属于同一血清型的IBV毒株归为同一类群。这些结果表明，本文所述的RT-PCR-RFLP系统可作为一种快速且经济的工具，用于区分IBV毒株。
