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Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco.

ABSTRACT: BACKGROUND:A rapid, sensitive, and specific molecular method for the diagnosis of infectious bronchitis virus (IBV) infection is important in curbing infectious bronchitis outbreaks in Morocco and other countries. METHODS:In this study, an easy-to-perform SYBR green I real-time reverse transcriptase polymerase chain reaction (RT-PCR) targeting the nucleocapsid gene of IBV was developed and compared with conventional agarose gel-based RT-PCR for the detection of IBV infection. RESULTS:We found that the SYBR green I real-time RT-PCR was at least 10 times more sensitive than the agarose gel electrophoresis detection method. The assay exhibited high specificity for IBV infection. All negative controls, such as Newcastle disease virus, infectious bursal disease virus, and avian influenza virus, were not detected. CONCLUSION:The SYBR green I real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for diagnosis and control of infectious bronchitis outbreaks in Morocco. The test is a valuable and useful method as a routine assay for diagnosis of clinical IBV infection in commercial chickens.

SUBMITTER: Fellahi S

PROVIDER: S-EPMC4841946 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco.

Fellahi Siham S El Harrak Mehdi M Kuhn Jens H JH Sebbar Ghizlane G Bouaiti El Arbi el A Khataby Khadija K Fihri Ouafae Fassi OF El Houadfi Mohammed M Ennaji My Mustapha MM

BMC research notes 20160422

<h4>Background</h4>A rapid, sensitive, and specific molecular method for the diagnosis of infectious bronchitis virus (IBV) infection is important in curbing infectious bronchitis outbreaks in Morocco and other countries.<h4>Methods</h4>In this study, an easy-to-perform SYBR green I real-time reverse transcriptase polymerase chain reaction (RT-PCR) targeting the nucleocapsid gene of IBV was developed and compared with conventional agarose gel-based RT-PCR for the detection of IBV infection.<h4>R ...[more]

PMID: 27106608

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Project description: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.

Dataset Information

Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco.

Publications

Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco.

Similar Datasets

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