Establishment and application of a multi-target detection method for porcine respiratory viruses based on MALDI-TOF MS

Abstract:

[Objective] To establish a high-throughput multi-target technique based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of porcine pseudorabies virus (PRV), porcine circovirus types 2 (PCV-2) and 3 (PCV-3), African swine fever virus (ASFV), and porcine bocavirus group 1 (PBoV-G1), group 2 (PBoV-G2) and group 3 (PBoV-G3). [Methods] The primers for different pathogens and corresponding single-base extension probes were designed based on the conserved sequences in the genes of the seven pathogens. After the optimization of primer concentrations and reaction conditions, the specificity, sensitivity, and repeatability of the method were analyzed. Finally, this MALDI-TOF MS method was used to detect the pathogens in clinical samples and porcine products. [Results] The established method only produced target peaks in the detection of specific virus-positive samples, and there was no cross-reaction with other pathogens, which indicated that the method had good specificity for the detection of the seven target viruses. The repeatability test results showed that the positive coincidence rate of each virus in high, medium, and low concentrations was ≥98.0% within batches and ≥98.3% between batches, indicating that the method had high repeatability. The low limits of detection of the established method for the seven pathogens in the system were within the range of 8.65-26.27 copies/μL, which was comparable to that of the real-time fluorescence quantitative PCR (RT-qPCR). The established MALDI-TOF MS method was used to detect 100 tissue, feed, and pork samples, in which 39 samples were detected with two or more mixed infections, including five samples simultaneously positive for five pathogens. In addition, eight samples of artificial contamination with recombinant adenovirus carrying ASFV-p72 gene were tested, which showed ASFV-positive results. The coincidence rates of the results obtained by MALDI-TOF MS and RT-qPCR for the detection of different pathogens were as high as 94.4%-100%. [Conclusion] The method established based on MALDI-TOF MS for the detection of multiple porcine respiratory DNA viruses in this study provides a sensitive, specific, high-throughput and multi-target detection technique for rapid surveillance and differential diagnosis of porcine diseases, facilitating the import and export animal quarantine.