Further development and use of a molecular serotype identification test for infectious bronchitis virus
Further development and use of a molecular serotype identification test for infectious bronchitis virus
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DOI：
10.2307/1592449
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发表时间：
1997-01-01
期刊：
影响因子：
1.4
通讯作者：
Hilt, DA
中科院分区：
文献类型：
--
作者：
Jackwood, MW;Yousef, NMH;Hilt, DA
在此之前，我们开发了一种快速的传染性支气管炎病毒（IBV）的血清型鉴定试验，利用逆转录-聚合酶链反应（RT-PCR）和限制性片段长度多态性分析。RT-PCR用于从从鸡蛋中生长的病毒提取的RNA扩增S1基因。PCR产物限制酶消化和电泳用于确定病毒的血清型。这项研究有三个目的。首先，我们用改良的5' PCR引物，改变了我们的快速血清型鉴定方法，并从实验接种了阿肯色州或马萨诸塞州41型IBV的无特异病原的来航鸡气管拭子中扩增了IBV的S1基因。气管拭子中IBV的直接扩增消除了在鸡蛋中分离病毒的需要。其次，我们试图在尿囊液中扩增灭活的IBV，这可能使我们能够获得和确定来自美国以外的分离株的血清型。用福尔马林（0.1%终浓度）灭活的病毒不能通过RT-PCR方法扩增，但热灭活的IBV（56 ℃，15分钟）成功地扩增。第三，我们通过合成克隆的截短的S1基因的RNA径流转录物开发了用于RT-PCR测试的内部对照。将截短的S1 RNA转录物加入到RT-PCR反应中，扩增出1031-bp的产物，该产物可以与来自病毒RNA的共扩增的S1基因区分开。内部RNA对照降低了RT-PCR检测中获得假阴性结果的可能性。
Previously, we developed a rapid serotype identification test for infectious bronchitis virus (IBV) that utilizes the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism analysis. The RT-PCR is used to amplify the S1 gene from RNA extracted from the virus grown in eggs. Restriction enzyme digestion and electrophoresis of that PCR product is used to determine the serotype of the virus. The purpose of this study was threefold. First, using a modified 5' PCR primer, we altered the procedures of our rapid serotype identification test and amplified the S1 gene of IBV in tracheal swabs collected from specific-pathogen-free leghorn chickens experimentally inoculated with the Arkansas or Mass 41 serotypes of IBV. Direct amplification of IBV in tracheal swabs eliminates the need to isolate the virus in eggs. Second, we attempted to amplify inactivated IBV in allantoic fluid, possibly allowing us to obtain and determine the serotype of isolates originating from outside the U.S.A. Virus inactivated by formalin (0.1% final concentration) could not be amplified by the RT-PCR procedure, but heat-inactivated IBV (56 C for 15 min) was successfully amplified. Third, we developed an internal control for the RT-PCR test by synthesizing RNA runoff transcripts of a cloned truncated S1 gene. The truncated S1 RNA transcripts were added to the RT-PCR reaction and a 1031-bp product was amplified, which could be distinguished from the coamplified S1 gene from viral RNA. The internal RNA control reduces the possibility of obtaining false-negative results in the RT-PCR test.