Specific Detection of African Swine Fever Virus Variants: Novel Quadplex Real-Time PCR Assay with Internal Control

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Animal Disease Control

Background:

• African swine fever (ASF) is a devastating viral disease impacting the global swine industry.
• Emerging African swine fever virus (ASFV) variants with genomic deletions challenge current control strategies.
• Accurate detection of these variants is crucial for effective disease management.

Purpose of the Study:

• To develop a novel, sensitive, and reliable quadplex real-time PCR assay.
• To detect ASFV variants with deletions in key genes (I177L, EP402R, MGF360-14L).
• To provide a tool for rapid and accurate ASF surveillance and control.

Main Methods:

• Designed a quadplex real-time PCR assay targeting conserved regions of ASFV.
• Incorporated a porcine beta-actin (ACTB) internal control to prevent false negatives.
• Validated the assay using spike-in tests, diverse sample panels, and cross-reactivity testing.

Main Results:

• The assay demonstrated high sensitivity with a limit of detection of 1-10 plasmid copies or 0.1 TCID50.
• 100% specificity was confirmed across various sample types, including field and experimentally infected pigs.
• No cross-reactivity was observed with other common swine viruses.

Conclusions:

• The developed quadplex real-time PCR assay is a robust tool for detecting ASFV variants.
• This assay facilitates timely identification and mitigation of ASF outbreaks.
• It supports enhanced surveillance and control efforts against emerging ASFV strains.