Development of a multiplex PCR for detection of avian adenovirus, avian reovirus, infectious bursal disease virus, and chicken anemia virus.

A multiplex polymerase chain reaction (mPCR) was developed and optimized for the simultaneous detection and differentiation of avian reovirus (ARV), avian adenovirus group I (AAV-I), infectious bursal disease virus (IBDV), and chicken anemia virus (CAV). Four sets of specific oligonucleotide primers were used in this test for ARV, AAV-I, IBDV, and CAV. The mPCR DNA products were visualized by gel electrophoresis and consisted of fragments of 365 bp for IBDV, 421 bp for AAV-I, 532 bp for ARV, and 676 bp for CAV. The mPCR assay developed in this study was found to be sensitive and specific. Detection of PCR-amplified DNA products was 100 pg for both CAV and IBDV, and 10pg for both ARV and AAV-I and this mPCR did not amplify nucleic acids from the other avian pathogens tested. The mPCR demonstrated similar sensitivity in tests using experimental fecal cloacal swab specimens that were spiked with ARV, AAV-1, IBDV, and CAV, and taken from specific pathogen free (SPF) chickens. This mPCR detected and differentiated various combinations of RNA/DNA templates from ARV, AAV-I, CAV, and IBDV without reduction of amplification from feces.