A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale

SIMPLE SUMMARY: Infectious respiratory diseases in poultry can be induced by viruses, bacteria, mycoplasmas, and fungi in single or mixed infections. Multifactorial infections require multiplex screening for the pathogens of concern. To meet this demand, gel-based multiplex PCR assays have been regularly developed and widely applied to simultaneously detect ten common respiratory pathogens (viruses and bacteria) in chickens. However, to date, there are no available gel-based multiplex PCR assays for the co-detection of Ornithobacterium rhinotracheale and infectious laryngotracheitis virus. In this study, we successfully developed and applied a new multiplex PCR method to detect these two pathogens in field samples. ABSTRACT: To date, many fluorescence- and gel-based multiplex polymerase chain reaction (PCR) assays have been developed for the simultaneous detection of multiple infectious agents of respiratory disease in poultry. However, PCR assays are not available for other important emerging respiratory bacteria, such as Ornithobacterium rhinotracheale (ORT). We aimed to fill this gap by establishing a new duplex PCR method for the simultaneous detection of infectious laryngotracheitis virus (ILTV) and ORT. Multiplex primer design software was used to select the compatible multiplex primer pairs. It was determined that an annealing temperature of 65 °C and an initial concentration of 2.5 pmol/µL for each primer set were the most suitable conditions for multiplex PCR. The assay was confirmed to be specific, as it only detected the target pathogens, even in the presence of six non-target agents. The limit of detection was up to 10(3) copies/µL of template DNA for both ILTV and ORT. In the screening of 304 field samples, 23, 88, and 44 were positive for both ILTV and ORT, solely for ILTV, and solely ORT, respectively.