Development of a duplex LNA-TaqMan real-time quantitative PCR for differential detection of virulent and attenuated strains of Muscovy duck-origin goose parvovirus.

Abstract

To establish a real-time quantitative PCR method for detecting and differentiating virulent and attenuated strains of Muscovy duck-origin goose parvovirus (MDGPV), a pair of common primers and two specific locked nucleic acid (LNA)-TaqMan probes targeting the conserved VP1 gene region were designed and synthesized based on MDGPV genome sequences from GenBank. By labeling the probes with distinct fluorophores and optimizing the reaction conditions, the optimal primer-probe combination was identified, and an LNA-TaqMan based quantitative PCR differentiation method was developed. The results demonstrated that this assay could specifically detect both virulent and attenuated MDGPV strains without cross-reactivity with other waterfowl viruses. The method exhibited high sensitivity, with a detection limit of 6.1 × 10copies/μL for both the virulent and attenuated strains. The method showed good reproducibility, with a coefficient of variation of less than 3 %. The detection results for the clinical samples were consistent with the sequencing analysis. These findings indicate that the established duplex LNA-TaqMan real-time quantitative PCR method is suitable for the differential detection of MDGPV virulent and attenuated strains in clinical samples, providing an effective technical tool for the control and eradication of MDGPV.