Multiplex detection and application of MALDI-TOF NAMS for porcine diarrheal pathogens

ABSTRACT Porcine diarrheal diseases caused by mixed viral and bacterial infections pose significant challenges to swine health and production. Rapid and accurate identification of key pathogens, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine bocavirus (PBoV), hepatitis E virus (HEV), and Salmonella , remains a challenge due to overlapping clinical symptoms and co-infections. The zoonotic potential of HEV and PBoV also highlights the need for reliable, high-throughput detection methods. We developed a multiplex Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Nucleic Acid Mass Spectrometry (MALDI-TOF NAMS) assay for the simultaneous detection of eight major porcine gastrointestinal pathogens. The method utilizes a multiplex PCR approach and a single-base extension strategy to improve sensitivity and specificity, and the extension products are displayed by mass spectrometry. The analytical performance of the assay was assessed by evaluating the limits of detection (LoD), analytical specificity, and repeatability. Additionally, we validated its diagnostic accuracy using 242 clinical samples, comparing the results with real-time quantitative PCR (qPCR) as the reference method. The MALDI-TOF NAMS assay demonstrated high analytical specificity, with no cross-reactivity to non-target pathogens. The limit of detection (LoD) ranged from 12.20 to 33.59 copies/μL. The intra- and inter-batch reproducibility assessments showed a 100% detection rate across high, medium, and low concentrations (20/20 per concentration, 60/60 total). Validation using 242 clinical samples demonstrated a 98.3% sensitivity and 99.5% specificity compared to qPCR, with an overall concordance rate of 96.2%. The MALDI-TOF NAMS assay provides a sensitive and high-throughput method for the detection and genotyping of major swine diarrhea pathogens. This method performs well in mixed infections and allows for expansion of pathogen species, making it an important addition to traditional diagnostic methods. IMPORTANCE Porcine diarrheal diseases are a major threat to the swine industry, often caused by multiple viruses and bacteria infecting animals at the same time. Fast and accurate detection of these pathogens is crucial to prevent outbreaks, reduce economic losses, and protect public health, especially given the potential of some pathogens to infect humans. This study introduces a new method that uses nucleic acid mass spectrometry technology to quickly and accurately detect eight major pathogens from pig samples, such as feces, blood, or tissue. Unlike traditional tests that often detect one pathogen at a time, this method can screen for many at once, even in complex cases of mixed infections. It is sensitive, reliable, and can handle large numbers of samples efficiently. This tool offers farmers, veterinarians, and disease control agencies a faster and more effective way to monitor pig health and respond to outbreaks before they spread.