Establishment and application of multiplex PCR for detection of three immunosuppressive viruses in waterfowl
Avian influenza virus H9 subtype(AIV-H9),goose circovirus(GoCV)and muscovy duck re-ovirus(MDRV)are common waterfowl pathogens.These viruses can easily cause immunosuppressive dis-eases in waterfowl,and mixed infections often occur.The specific primers were designed for the gene sequences of AIV-H9,GoCV and MDRV,respectively.After optimizing the reaction conditions of multiplex PCR,the method for three viruses was established.Then,the specificity,sensitivity and repeatability were verified.The results showed that the established triple PCR assay could specifically amplify the target fragments of AIV-H9,GoCV and MDRV,and it had no cross-reaction with novel-goose astrovirus,duck enteritis virus,goose parvovirus and fowl adenovirus serotype 4.Sensitivity assay showed that the minimum concentrations were 2.93 × 104 copies/μL,2.5 × 102 copies/μL and 3.0 × 106 copies/μL,re-spectively.The triple PCR was used to detect 30 cl inical samples,the positive rates of MDRV was 6.7％,GoCV and AIV-H9 were 36.7％and 26.7％,respectively.The coincidence rate of triple PCR and single PCR was 100％.The results showed that the triple PCR method was specific,sensitive and reproducible,it could be used for epidemiological investigation and clinical detection of the three viruses.
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