Development and application of a quadruplex real-time fluorescence quantitative PCR assay for four porcine digestive pathogens.

INTRODUCTION

, , , and are the primary pathogens responsible for gastrointestinal diseases in pigs, posing a significant threat to the health and productivity of pig production systems. Pathogen detection is a crucial tool for monitoring and managing these infections.

METHODS

We designed primers and probes targeting the gene of , the 23S gene of , the gene of , and the gene of . We developed a quadruplex TaqMan real-time quantitative PCR assay capable of simultaneously detecting these four pathogens.

RESULTS

This assay demonstrated high sensitivity, with detection limits of 100 copies/μL for the recombinant plasmid standards pEASY-23S rRNA, pEASY-aspA, and pEASY-, and 10 copies/μL for pEASY-. The standard curves exhibited excellent linearity (R values of 0.999, 0.999, 1, and 0.998, respectively) and high amplification efficiencies (93.57%, 94.84%, 85.15%, and 81.81%, respectively). The assay showed high specificity, with no cross-reactivity detected against nucleic acids from Streptococcus suis, porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), , , , porcine delta coronavirus (PDCoV), porcine group A rotavirus (GARV), and porcine teschovirus (PTV). The assay also exhibited excellent repeatability, with inter- and intra-assay coefficient of variation (CV) ranging from 0.15% to 1.12%. High concentrations of nucleic acids did not interfere with the detection of low concentrations, ensuring robust performance in complex samples. Among 263 diarrhoeic samples, the assay detected in 23.95%, in 26.24%, in 33.84%, and in 22.43%.

DISCUSSION

This quadruplex TaqMan qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of , , , and in pigs.