Establishment and Evaluation of Triple-primer Duplex PCR Method for Differential Detection of Muscovy duck-origin goose parvovirus and Muscovy duck parvovirus
WANG Shao1,*, LIU Hong-Wei1,2,*, CHENG Xiao-Xia1, ZHU Xiao-Li1, JIANG Dan-Dan1, XIAO Shi-Feng1, CHEN Shao-Ying1,**, CHEN Shi-Long1,**
1 Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350013, China; 2 College of Animal Sciences, Fujian Agricultural and Forestry University, Fuzhou 350002, China
Abstract：Muscovy duck-origin goose parvovirus (MDGPV) is a novel waterfowl parvovirus generated by genetic recombination of Classical Goose parvovirus (cGPV) and Muscovy Duck parvovirus (MDPV). It is one of the main pathogens causing severe diarrhea and exudative enteritis in young Muscovy ducks (Cairina moschata), leading to a decline in their immunity and an increase in morbidity and mortality. The study aimed to establish a three-primer PCR method for simultaneous detection and differentiation of MDGPV and MDPV and monitor the current epidemic situation of the 2 viruses. Based on the recombination characteristics of the viral capsid protein (VP1) gene of MDGPV and MDPV published in GenBank, 3 differential primers were designed and synthesized using the Oligo 7.0 software. Two primers were combined to form 2 sets of PCR reaction systems for amplification, with the target fragment sizes of 493 bp (MDGPV) and 827 bp (MDPV). Standard positive recombinant plasmids of MDGPV and MDPV were constructed as reference materials for the amplification test. The results showed that this study successfully established a three-primer duplex PCR discrimination method capable of simultaneously detecting MDGPV and MDPV. The three-primer duplex PCR method had good repeatability, high specificity and sensitivity, with the minimum detection limits for MDGPV and MDPV being 99.6 and 96.1 copies/μL of total DNA, respectively. The detection results for 102 clinical samples suspected of being infected with waterfowl Parvovirues were as follows: the positive detection rate of MDGPV was 31.37% (32/102), the positive detection rate of MDPV was 7.84% (8/102), the positive detection rate of mixed infection was 1.96% (2/102), which were consistent with the results of SYBR Green Ⅰ-based PCR detection and virus isolation for diagnosing waterfowl Parvovirus infections. In summary, the three-primer PCR diagnostic method established in this study has high specificity, good sensitivity, and is a valuable addition to the detection methods for avian Parvovirus diseases. It is helpful for the clinical diagnosis and epidemiological surveillance of MDGPV and MDPV.
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