MALDI-TOF Nucleic Acid Mass Spectrometry for Simultaneously Detection of Fourteen Porcine Viruses and its Application

Background: Porcine respiratory and digestive diseases pose significant challenges in modern pig farming, often arising from mixed infections involving various pathogens. Current methods for detecting viral porcine pathogens have notable limitations in simultaneously identifying multiple pathogens. To address this issue, our study introduces a novel methodology that combines single-base extension PCR with matrix-assisted laser desorption/ionization time-of-flight nucleic acid mass spectrometry (MALDI-TOF NAMS). Results: Our approach accurately simultaneously identified 14 critical porcine viruses, including porcine circovirus types 1 to 3, porcine bocaviruses groups 1 to 3, African swine fever virus, pseudorabies virus, porcine parvovirus, torque teno sus virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. The low limit of detection for multiplex identification ranges from 13.54 to 1.59 copies/μL. Inter- and intra-assay stability was found to be ≥98.3%. In a comprehensive analysis of 108 samples, the assay exhibited an overall compliance with qPCR results of 97.88%. Conclusions: The developed MALDI-TOF NAMS assay exhibits high sensitivity, specificity, and reliability in detecting and distinguishing a wide spectrum of porcine viruses in complex matrix samples. This underscores its potential as an efficient diagnostic tool for disease surveillance and control in the pig industry.