Development and Application of a Multiplex Reverse Transcription- Polymerase Chain Reaction for Avian Viral Respiratory Agents

Abstract

Respiratory tract infections are critical in the poultry industry. Separated reverse transcription-polymerase chain reaction (RT-PCR) assays for different avian diseases require several reactions. A multiplex RT-PCR assay that detects avian viral respiratory pathogens in the same reaction requires less time for clinical practice. Four sets of specific oligonucleotide primers for infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) were used in this study. Both multiplex RT-PCR and single RT-PCR resulted in expected products. The sensitivity limits of this multiplex RT-PCR were 3.5ng, 0.14ng, 0.4ng, and 0.36ng for ILTV, IBV, NDV, and AIV, respectively. Among the tested samples, seventy-seven percent of the 22 virus isolation-positive samples showed positive by genome detection of homogenates. In addition, ninety-one percent of the 64 virus isolation-negative samples showed negative by genome detection of homogenates. Thus, the multiplex RT-PCR was able to detect pathogens in clinical samples for rapid identification and differential diagnosis of poultry viral respiratory infections.