Development and Application of a Quadruplex Real-time Fluorescence Quantitative PCR Assay for Four Porcine Digestive Pathogens

Overview

Journal Front Cell Infect Microbiol

Specialties Cell Biology
Infectious Diseases
Microbiology

Date 2024 Dec 11

PMID 39660284

Authors

Haojie Wang

Yue Sun

Jianxing Chen

Wei Wang

Haibo Yu

Caixia Gao

Tongqing An

Yue Wang

Hongyan Chen

Liangquan Zhu

Zhimin Jin

Changqing Yu

Changyou Xia

He Zhang

Affiliations

Soon will be listed here.

Abstract

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.

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