Development and preliminary application of a multiplex PCR method for the detection of goose parvovirus and goose astrovirus 1 and 2

Abstract

Goose astrovirus 1 (GAstV1), goose astrovirus 2 (GAstV2), and goose parvovirus (GPV) infections have significant negative effects on the goose farming industry. A rapid and accurate multiplex PCR method for simultaneous detection of these 3 goose viruses is needed. We designed and synthesized specific primers targeting the conserved regions of GAstV1, GAstV2, and GPV. and optimized reaction conditions to assess specificity and sensitivity. The optimal amplification condition was an annealing temperature of 54.5°C for 35 cycles. Specific bands were detected at 315 bp (GAstV1), 609 bp (GAstV2), and 1,405 bp (GPV). No cross-amplification occurred with other common goose pathogens, including avian influenza A virus H9, Newcastle disease virus, duck Tembusu virus, fowl adenovirus 4, goose reovirus, and goose circovirus. The limit of detection (LOD) for GAstV1, GAstV2, and GPV was 1 × 10³ copies/μL, and the LOD for samples containing all 3 agents was 1 × 10⁴ copies/μL. Our results from testing 90 clinical samples were in 100% concordance with single PCR assays, suggesting that our multiplex PCR assay offers a reliable and efficient approach for the clinical detection of GAstV1, GAstV2, and GPV.