Development and evaluation of quadruplex droplet digital PCR assay for rapid detection of molecular markers associated with macrocyclic lactone resistance and susceptibility in Dirofilaria immitis

Due to climate change and human interventions, there has been an increase in D. immitis infections, underscoring the necessity for monitoring the spread and extent of resistance. In our prior research, we introduced a rapid test utilizing four predominant SNP markers at loci 15709 (SNP1), 30575 (SNP2), 21554 (SNP3), and 9400 (SNP7) linked to ML resistance. Our findings highlighted SNP1 and SNP2 as potent predictive markers, offering suitability for the rapid detection and monitoring of drug resistance. Therefore, we developed a cost-effective test using droplet digital PCR (ddPCR) technology to perform a quadruplex assay to assess alternate allele frequency. Our assay can identify both SNP1 and SNP2 wildtype and mutant targets in a single sample. We tested the performance of our 4-plex assay on 8 laboratory-maintained isolates, including Missouri (MO), Berkeley, JYD-34, Metairie-2014, Yazoo-2013, GA-2, GA-3, and Big Head, and further validated the sensitivity using clinical isolates from the United States. Our assay demonstrated consistent results compared to the standard MiSeq sequencing and ddPCR duplex assay. Notably, we showcased the utility of ddPCR for direct genotyping of D. immitis infected whole blood from two well-characterized isolates, MO and JYD-34. This approach streamlined the assay workflow and lowered costs, thus enhancing affordability for larger genotyping studies.