Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens.
Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens.
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DOI：
10.1016/j.jviromet.2006.07.018
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发表时间：
2006-12
影响因子：
3.1
通讯作者：
Jackwood MW
中科院分区：
文献类型：
--
作者：
Callison SA;Hilt DA;Boynton TO;Sample BF;Robison R;Swayne DE;Jackwood MW
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疾病病原是非常重要的，它们在发病的早期阶段可能非常相似。在这项研究中，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IBV的早期和快速检测。该试验扩增IBV基因组5 ' -UTR中143 bp的产物，每次反应的检测和定量限为100个模板拷贝。检测的所有15种IBV菌株以及两种土耳其冠状病毒株均被扩增，而检测的其他病原体均未呈阳性。用1329份气管拭子样本完成了该检测方法的评估。实时RT-PCR法检测IBV抗体阴性的禽类680份样本，结果显示IBV阴性。我们对提交给两个不同诊断实验室的229份气管拭子进行了检测，发现79.04%的气管拭子通过实时RT-PCR检测呈IBV阳性，而只有27.51%的样本通过参考标准检测病毒分离检测呈阳性。我们还在6个不同的时间点收集了120份来自实验感染不同剂量IBV的鸟类的气管拭子，发现接种后5天气管平台的病毒载量与给药剂量无关。此外，在接种后14天观察到病毒剂量与病毒载量呈反比关系。最后，我们测试了300个气管拭子样本，这些样本来自一群在野外喷洒IBV疫苗的商业肉鸡。在接种后3、7和14天感染IBV疫苗的鸟类比例分别为58%、65%和83%，这表明只有略多于一半的鸟类最初被感染，然后疫苗随后传播给了群中的其他鸟类。这一观察结果意义重大，因为具有高突变率的冠状病毒在发生鸟对鸟传播时可以恢复致病性。本文描述的实时RT-PCR检测可用于快速区分IBV与其他呼吸道病原体，这对控制这种高传染性病毒具有重要意义。该测试具有极高的敏感性和特异性，可用于定量临床样品中的病毒基因组RNA。
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.
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影响因子：
1.4
作者：
Jackwood, MW;Yousef, NMH;Hilt, DA
通讯作者：
Hilt, DA
影响因子：
5
作者：
Lee, CW;Jackwood, MW
通讯作者：
Jackwood, MW
影响因子：
2.8
作者：
NAQI, SA;KARACA, K;BAUMAN, B
通讯作者：
BAUMAN, B
影响因子：
3.3
作者：
KARACA, K;NAQI, S
通讯作者：
NAQI, S
影响因子：
1.4
作者：
KARACA, K;NAQI, S;GELB, J
通讯作者：
GELB, J