Development and application of a multiplex PCR method for simultaneous detection of waterfowl parvovirus, duck enteritis virus and goose astrovirus.

Waterfowl parvovirus, duck enteritis virus and goose astrovirus have become serious pathogens in waterfowl farming. Co-infections occasionally occur, and as a result, it is much harder to rapidly and simultaneously identify several pathogens using conventional PCR. According to the characteristics of the goose parvovirus (GPV) and muscovy duck parvovirus (MDPV) genome sequences, a universal PCR primer was designed using Rep1 as the target gene. The specific detection primers were designed based on the specific conserved regions of UL54 of the duck enteritis virus (DEV) gene and ORF1a of the goose astrovirus (GAstV) gene. The PCR reaction system and conditions were optimized, and the optimal annealing temperature was found to be 56.2 ℃. The volume ratio of the GPV-MDPV, GAstV and DEV primers (20 μM) was 1:4:5. The established multiplex PCR detection method can simultaneously detect GPV, MDPV, DEV and GAstV within one reaction, and be negative for duck Tembusu virus, muscovy duck reovirus, duck hepatitis A virus type 3 and duck circovirus. The method with excellent sensitivity, specificity and repeatability was successfully applied to clinical samples, it is a useful platform for identifing co-infections of GPV, MDPV, DEV and GAstV in waterfowl. © King Abdulaziz City for Science and Technology 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.