Related Experiment Video
Updated: Aug 3, 2026
Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR
Published on: June 26, 2016
Droplet digital PCR as a tool to detect resistant isolates of Dirofilaria immitis
Sohini Kumar1, Roger K Prichard1, Thavy Long1
1Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, H9X3V9, QC, Canada.
Abstract:
Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance.
Insights
A new droplet digital PCR method rapidly detects genetic markers for macrocyclic lactone resistance in canine heartworm (Dirofilaria immitis). This tool aids in identifying resistant parasites and monitoring drug resistance in canine populations.
Area of Science:
- Veterinary Parasitology
- Molecular Diagnostics
- Antiparasitic Drug Resistance
Background:
- Canine heartworm disease prevention relies on macrocyclic lactones (MLs).
- Emerging drug resistance in Dirofilaria immitis poses a significant threat to effective prevention.
- Current methods for detecting resistance are limited, hindering accurate diagnosis and monitoring.
Purpose of the Study:
- To develop a rapid and cost-effective diagnostic assay for detecting genetic markers associated with ML resistance in D. immitis.
- To validate a droplet digital PCR (ddPCR) protocol for identifying single nucleotide polymorphisms (SNPs) linked to drug resistance.
- To assess the utility of ddPCR for distinguishing between susceptible, resistant, and mixed-genotype D. immitis isolates.
Main Methods:
- Development of a specific droplet digital PCR (ddPCR) protocol using custom primers and hydrolysis probes.
- Targeting ten previously identified single nucleotide polymorphisms (SNPs) associated with ML resistance.
- Comparison of ddPCR results with MiSeq sequencing data and phenotypic resistance profiles of D. immitis isolates.
Main Results:
- The ddPCR assay accurately detected and quantified SNPs in both susceptible (Missouri) and resistant (JYD-34) D. immitis isolates.
- SNP1 and SNP2 were identified as particularly predictive markers for rapid detection of ML resistance.
- The assay successfully identified isolates with susceptible, resistant, and mixed genotypes, indicating its ability to detect heterogeneous parasite populations.
Conclusions:
- Droplet digital PCR offers a sensitive, rapid, and potentially more accessible method for detecting genetic markers of ML resistance in D. immitis.
- This ddPCR assay can differentiate between infections with susceptible and resistant parasites, including mixed infections.
- The developed ddPCR protocol is a valuable tool for epidemiological surveys, individual isolate assessments, and monitoring the development of drug resistance in canine heartworm.
