Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus.

The H9N2 subtype of avian influenza virus (AIV) and infectious bronchitis virus (IBV) are important avian viruses that cause respiratory symptoms in poultry, and can form mixed infections. In this study, primers and probes were designed based on the HA gene of H9N2 and the 5' noncoding region of IBV, respectively, and a fluorescent quantitative RT-PCR assay was established for simultaneous detection of these two pathogens. The reaction system and conditions were optimized. The method only detected AIV subtype H9N2 and IBV and no other viruses, confirming its high specificity. The assay detected 13.5 copies/μL and 1.66 copies/μL of H9N2 and IBV in clinical samples, respectively. The coefficients of variation for intra- and interassay repeatability were < 3%. The established method was used to analyze 254 clinical samples (oropharyngeal and cloacal swabs) from Hubei Province, China; 98.82% were positive for both pathogens. In summary, a duplex fluorescent quantitative RT-PCR method capable of simultaneously detecting AIV subtype H9N2 and IBV was established. It is specific, sensitive, and reproducible, and can be used for diagnosis of a variety of clinical samples. It provides a technological means for the rapid and simultaneous detection of both pathogens, and thus can facilitate clinical diagnosis and epidemiological investigations.