A MALDI-TOF MS system for simultaneous detection of six RNA viruses causing porcine respiratory diseases

Abstract:

[Objective] To establish a multi-target method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), and swine influenza virus (SIV), and the detection of the general type, H1 type and H3 type of SIV. [Methods] We designed six pairs of primers and corresponding extension probes according to the conserved sequences of six pathogens and conducted single reaction tests. The primer concentrations and the reaction conditions were optimized for the established method. After examination of the specificity, repeatability, and sensitivity, the MALDI-TOF MS method was used to detect clinical samples and pig-derived products, and the results were compared with those obtained by fluorescence quantitative PCR. [Results] The established method only generated peaks corresponding to the positions of the target viruses, and other common pathogens causing swine diseases did not appear in the mass spectra, which indicated that the system had good specificity. The in-batch positive coincidence rate of each virus at high, medium, and low concentrations was 100%, and the inter-batch coincidence rate was ≥ 96.7%, indicating that the system had good repeatability. The minimum limit of detection for each virus in the system varied within 6.73–21.25 copies/μL. We then used the MALDI-TOF MS method to detect 124 tissue, feed, and pork samples. A total of 12 positive samples were detected, including nine PRRSV positive samples and three SIV positive samples (two SIV-H1 positive samples and one SIV-H3 positive sample). We then compared the above results with the results from fluorescence quantitative PCR, and found that the total coincidence rate of the two methods for detection of each pathogen was 99.2%–100%. [Conclusion] A method for the simultaneous detection of six RNA viruses causing porcine respiratory diseases was established based on MALDI-TOF MS. This sensitive and specific high-throughput multi-target detection method facilitates the surveillance and rapid identification of related diseases in the import and export animal quarantine.