Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Microbiology

Background:

• Sensitive detection of pathogenic microorganisms is critical for disease control.
• African Swine Fever Virus (ASFV) poses a significant threat to swine populations globally.
• Existing diagnostic methods require improvement in sensitivity and specificity.

Purpose of the Study:

• To develop a highly sensitive TaqMan real-time quantitative PCR (qPCR) method for ASFV detection.
• To investigate a multi-probe strategy to enhance detection sensitivity.
• To validate the assay's performance using clinical samples and compare it with established methods.

Main Methods:

• Design and optimization of four probes targeting the ASFV *B646L* gene.
• Development of a single TaqMan qPCR assay incorporating the multi-probe strategy.
• Ultrafiltration for concentrating viral nucleic acids from water samples.
• Cross-reactivity testing with common swine viruses and validation with 173 clinical samples.

Main Results:

• The established TaqMan qPCR assay detected as low as 0.5-5 copies/μl of the ASFV gene.
• Ultrafiltration enabled sensitive detection of trace viral nucleic acids.
• No cross-reactivity was observed with other common swine viruses.
• 100% coincidence rate with the WOAH-recommended method in clinical sample detection.

Conclusions:

• A highly sensitive and specific TaqMan qPCR assay for ASFV detection was successfully established.
• The multi-probe strategy significantly enhances detection sensitivity for trace pathogenic microorganisms.
• This integrated strategy offers a valuable tool for sensitive ASFV diagnostics and potentially other pathogens.