Callison Scott A, Hilt Deborah A, Boynton Tye O, Sample Brenda F, Robison Robert, Swayne David E, Jackwood Mark W
Department of Population Health, Poultry Diagnostic and Research Center, 953 College Station Road, University of Georgia, Athens, GA 30602, USA.
J Virol Methods. 2006 Dec;138(1-2):60-5. doi: 10.1016/j.jviromet.2006.07.018. Epub 2006 Aug 28.
It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.
快速区分传染性支气管炎病毒（IBV）与高致病性禽流感病毒和外来新城疫病毒等病原体很重要，因为它们在发病初期可能极为相似。在本研究中，我们报告了一种使用Taqman标记探针的实时RT-PCR检测方法的开发和测试，用于早期快速检测IBV。该检测方法在IBV基因组的5'-UTR区域扩增出一个143bp的产物，每个反应的检测和定量限为100个模板拷贝。所检测的15株IBV以及两株火鸡冠状病毒株均被扩增，而其他检测的病原体均未检测为阳性。用1329份气管拭子样本完成了该检测方法的评估。从IBV抗体阴性鸡采集的总共680份样本经实时RT-PCR检测为IBV阴性。我们检测了提交给两个不同诊断实验室的229份气管拭子，通过实时RT-PCR发现79.04%的气管拭子IBV呈阳性，而作为参考标准检测的病毒分离法仅27.51%的样本呈阳性。我们还在六个不同时间点从经不同剂量IBV实验感染的鸡采集了总共120份气管拭子，发现无论接种剂量如何，接种后5天气管中的病毒载量达到平稳状态。此外，观察到接种后14天给予的病毒剂量与病毒载量之间呈反比关系。最后，我们检测了来自田间一群针对IBV进行喷雾免疫的商品肉鸡的300份气管拭子样本。接种疫苗后3天、7天和14天感染IBV疫苗的鸡的百分比分别为58%、65%和83%，这表明最初只有略多于一半的鸡被感染，随后疫苗传播给了鸡群中的其他鸡。这一观察结果很重要，因为冠状病毒突变率高，当发生鸡与鸡之间的传播时可能恢复致病性。本文所述的实时RT-PCR检测方法可用于快速区分IBV与其他呼吸道病原体，这对于控制这种高传染性病毒很重要。该检测极为灵敏且特异，可用于定量临床样本中的病毒基因组RNA。
