Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, H9X3V9, QC, Canada.
Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, H9X3V9, QC, Canada.
Int J Parasitol Drugs Drug Resist. 2023 Dec;23:10-18. doi: 10.1016/j.ijpddr.2023.07.002. Epub 2023 Jul 23.
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.
犬心丝虫病是由犬恶丝虫引起的，预防该病依赖于大环内酯类药物，但目前这类药物的耐药性令人担忧。虽然犬恶丝虫种群的耐药性与遗传多态性有关，但耐药机制仍不清楚。缺乏可靠的体外检测方法来检测耐药性是确认耐药性的一个限制。先前曾使用 MiSeq 平台在犬恶丝虫耐药分离株中对 10 个单核苷酸多态性（SNP）进行了临床验证。该技术虽然对研究很有用，但价格昂贵，无法在少量临床样本中快速检测这些标记物。我们开发了一种用于检测与 ML 耐药相关 SNP 的数字 PCR 协议。设计了包含野生型和突变型等位基因的特异性引物和水解探针，以从不同犬恶丝虫分离株的基因组 DNA 中扩增 SNP 靶标。确定了等位基因频率，并评估了 ddPCR 检测的适用性，并与 MiSeq 数据进行了比较。ddPCR 检测准确地检测到并定量了参考菌株中两个不同的等位基因，即 ML 敏感的密苏里州（MO）和 ML 耐药的 JYD-34，在先前确定的 SNP 位置。还确定了具有已知或假定敏感或耐药表型的其他分离株中 SNP 的存在。我们观察到 SNP1 和 SNP2 是更具预测性的标记物，似乎适合于快速检测和监测耐药性。我们的结果表明，ddPCR 可用于区分因实际遗传耐药性引起的感染与因敏感寄生虫感染，也可用于快速检测不仅具有 ML 敏感和耐药基因型的分离株，还可检测对应于含有 ML 敏感和耐药寄生虫混合种群的异质分离株的混合基因型的分离株。ddPCR 可能是一种有用的工具，用于进行调查或评估个体分离株的遗传耐药性证据或耐药性的发展。
