Fowl adenovirus (FAdV) infections in chickens have resulted in global economic losses in the poultry industry. Infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) infections lead to immunosuppression in chickens, and concomitant co- infection with FAdV usually produces severe and lethal infections. These co-infections are common occurrences on chicken farms and affect large number of chickens. Thus, a rapid, sensitive and specific diagnostic test for these viruses becomes a prerequisite to effective control and isolation measures. We developed a triplex nanoparticle-assisted PCR (nano-PCR) assay that can simultaneously detect these 3 viruses in a single assay tube using PCR primers directed at respective specific genes of each virus. The assay was specific for FAdVs, CAV and IBDV, and it did not amplify Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus or Marek's disease virus. The minimum detection limit was 27.2 femtogram (fg) for all three viruses and was 1000-fold more sensitive than multiplex PCR using identical primers. Screening of 69 clinical samples from 40 to 50 days old chickens with obvious lesions in liver using the nano-PCR compared with a multiplex PCR yielded identical results. Of the 69 samples, 13 were detected positive including 4 for FAdV, 4 for IBDV and 6 for CAV single virus infections, respectively, as well as 5 for FAdV/CAV, 2 for FAdV/IBDV and 3 for IBDV/CAV co-infections. The triple nano-PCR assay developed in our laboratory is a sensitive, specific and simple method that can be used for detection of FAdV, CAV and IBDV as single or mixed infections.
