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.

Siham Fellahi,Ghizlane Sebbar,Mehdi El Harrak,Khadija Khataby,Jens H Kuhn,My Mustapha Ennaji,El Arbi Bouaiti,Mohammed El Houadfi,Ouafae Fassi Fihri

doi:10.1186/s13104-016-2037-z

Abstract

BackgroundA 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.MethodsIn 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.ResultsWe 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.ConclusionThe 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.

Highlights

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
Reproducibility SYBR green I real‐time RT‐PCR The cDNA IBV Beaudette strain was used to extract the standard control genomic ribonucleic acid (RNA) that served as the source of cDNA template
Conventional gel‐based RT‐PCR The PCR targeted a sequence corresponding to a region of the IBV N protein

Summary

Introduction

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. Infectious bronchitis (IB), an acute, highly contagious viral upper respiratory disease of chickens, is one of the most economically significant diseases hampering the intensive poultry industry worldwide. IB affects chickens of all ages, causing respiratory, reproductive, and renal manifestations [1]. Control of IBV infection is primarily achieved through live attenuated vaccines, the IB is a disease that negatively impacts the poultry industry of developing countries. The causative agent of IB, infectious bronchitis virus (IBV), is a member of the species Avian coronavirus, genus Gammacoronavirus, family Coronaviridae [7]. The S protein contains two posttranslational subunits, S1 and S2 [9]

Methods

Results

Conclusion