Multiplex PCR Approach for Rapid African Swine Fever Virus Genotyping

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Genomic Epidemiology

Background:

• African swine fever virus (ASFV) poses a significant threat to the global pig industry due to its high mortality and widespread outbreaks.
• Traditional whole genome sequencing for ASFV tracing is time-consuming and expensive.
• Current genotyping relies on single PCR amplicon sequencing, which can be inefficient for rapid diagnostics.

Purpose of the Study:

• To develop and evaluate a cost-effective and time-efficient multiplex PCR method for ASFV genotyping.
• To assess the utility of Nanopore ligation-based amplicon sequencing for ASFV strain identification.
• To provide a versatile tool for rapid ASFV genotyping during disease outbreaks.

Main Methods:

• Development of a multiplex PCR assay using four established ASFV genotyping primer sets (B646L, E183L, B602L, I73R-I329L).
• Application of Nanopore ligation-based amplicon sequencing to multiplex PCR products.
• Testing the protocol on ASFV DNA from diverse biological matrices.

Main Results:

• The multiplex PCR protocol successfully genotyped ASFV DNA from various biological samples.
• The method accurately classified multiple ASFV genotypes and strains within a single PCR reaction.
• This approach significantly reduces the time and cost associated with ASFV genotyping.

Conclusions:

• Multiplex PCR combined with Nanopore sequencing offers a streamlined and economical solution for ASFV genotyping.
• This versatile method can be adapted by adding or modifying primer sets to meet regional needs.
• The optimized protocol can accelerate ASFV outbreak response and enhance disease surveillance efforts.