Development of multiplex oligonucleotide microarray for simultaneous detection of six swine pathogens

Area of Science:

• Veterinary Microbiology
• Molecular Diagnostics
• Animal Virology

Background:

• Accurate and rapid detection of multiple swine pathogens is crucial for disease control.
• Existing diagnostic methods may lack the capacity for simultaneous, high-throughput pathogen identification.

Purpose of the Study:

• To develop and evaluate a multiplex PCR technique for simultaneous detection of six major swine pathogens.
• To establish a high-throughput identification method for epidemic investigation and clinical diagnosis.

Main Methods:

• Designed six pairs of primers and probes targeting conserved sequences of APP, HPS, PRRSV, Mhp, PCV-2, and CSFV.
• Developed and optimized a multiplex PCR system and oligonucleotide microarray.
• Validated the assay using clinical blood samples and sequencing verification.

Main Results:

• Achieved high sensitivity for detecting individual pathogens, with limits as low as 5.8 × 10^2 copies/μL for APP.
• Demonstrated no cross-reactivity with nine other common swine pathogens.
• Identified single infections in 18.2% and mixed infections in 6.3% of 285 clinical samples.

Conclusions:

• The developed multiplex PCR technique is a sensitive, specific, and rapid method for simultaneous detection of six major swine pathogens.
• This technology offers a valuable tool for high-throughput screening, clinical diagnosis, and epidemiological surveillance of swine diseases.