Development of a Microplate Lectin-Capture RT-PCR (MLC-RT-PCR) for the Detection of Avian Infectious Bronchitis Virus

Maria de Fatima S. Montassier, Vanessa Mirabeli T. Piza, Cintia Hiromi Okino, Liana Brentano, Leonardo José Richtzenhain, Helio José Montassier

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Laboratory of Virology and Immunology–IMUNOVIR–Department of Veterinary Pathology of University of Sao Paulo State (FCAV–UNESP), Jaboticabal, Brazil.

DOI: 10.4236/aim.2013.33039                

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.

Keywords

Molecular Diagnosis; Infectious Bronchitis Virus; RNA Separation; Lectin-Capture

Conflicts of Interest

The authors declare no conflicts of interest.

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