Abstract
Rapid, sensitive and specific methods are necessary to confirm the diagnosis of outbreaks of avian infectious bronchitis virus (IBV) infection. The amplification of IBV genome by reverse transcription followed by polymerase chain reaction (RT-PCR) has been one of the most used methods for the detection of this virus in clinical samples. To reduce the time and the number of steps in the molecular diagnosis of IBV, we developed a sensitive and rapid detection method based on viral capture by a lectin (Concanavalin A—Con A) in the microplate wells, followed by RT-PCR to amplify the S1 gene. The detection limit of IBV was 103 EID50/ml for the amplification of 5’part of the S1 gene, and 104 EID50/ml for the amplification of full S1 gene. This technique was specific for IBV detection, and no amplified products were detected for other avian viral pathogens (bursal infectious disease virus, avian metapneumovirus and Newcastle disease virus). The MLC-RT-PCR was as sensitive as conventional RT-PCR, and virus isolation method for the detection of IBV in tissue samples collected from experimentally infected birds. The MLC-RT-PCR technique demonstrated a great potential for the rapid and specific diagnosis of IBV.
Highlights
Infectious bronchitis virus (IBV) is one of the major causes of economic losses in the poultry industry and can be result in respiratory disease, nephritis, and poor egg production and egg quality [1,2]
The IBV was effectively captured by the lectin Con A attached to the microplate wells, and after the addition of the buffer and RT reagents generated, after appropriate incubation, a cDNA which was amplified in the PCR, by primer sets 1 and 2 for S1 gene (Figure 1)
None of the other viruses non-related to IBV (Newcastle disease virus, avian metapneumovirus, infectious bursal disease virus) tested here showed any one of these amplified products (Figure 1)
Summary
Infectious bronchitis virus (IBV) is one of the major causes of economic losses in the poultry industry and can be result in respiratory disease, nephritis, and poor egg production and egg quality [1,2]. IBV is an important avian pathogen characterized by a worldwide distribution and many different variants of this virus are appearing continuously, despite the use of vaccines [4]. This virus is classified in the Gammacoronavirus Genus from the Coronaviridae Family [5]. The IBV contains a genome constituted by a single stranded RNA of positive polarity that consists of approximately 27 kb and codes for four main structural proteins: the spike glycoprotein (S), the small envelope (E) protein, the membrane glycoprotein (M) and the nucleocapside protein (N) [6]
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