Duplex real-time qPCR assay targeting β-tubulin SNPs for detection of albendazole resistance in Haemonchus contortus.

Haemonchus contortus (H. contortus), a major gastrointestinal parasitic nematode threatening ruminants worldwide, exhibits albendazole resistance that is primarily associated with single nucleotide polymorphisms (SNPs) at specific codons of the β-tubulin isotype I gene, particularly at codons 167 (F167Y), 198 (E198A), and 200 (F200Y). Among these, mutations at codons 198 and 200 are the most frequently detected in resistant isolates in China. To enable rapid and accurate resistance detection, this study aimed to develop a duplex real-time quantitative PCR (duplex qPCR) assay targeting SNPs at codons 198 and 200 of the β-tubulin gene for precise discrimination of albendazole resistance in H. contortus. Specific primers and TaqMan probes targeting codons 198 and 200 of the β-tubulin isotype I gene were designed to establish singleplex and duplex qPCR detection systems. The singleplex qPCR showed a detection limit of 1 × 101 copies/μL for the sensitive, 198-resistant, 200-resistant, and mixed genotypes, with high specificity and good repeatability (intra-assay coefficient of variation (CV) < 0.72%, inter-assay CV < 0.77%). Based on this, an optimized duplex qPCR was developed, achieving detection limits of 1 × 10 ³ copies/μL for the sensitive genotype and 1 × 101 copies/μL for the resistant genotype, with intra-assay and inter-assay CVs < 0.55%. This method effectively distinguished sensitive, resistant, and mixed infection samples. Application to 102 clinical samples identified 1 sensitive, 25 resistant, and 76 mixed infections. Sequencing of seven randomly selected samples produced results identical to the qPCR findings, further confirming the assay's sensitivity and accuracy. This study successfully established a rapid, sensitive, and specific duplex qPCR assay capable of accurately discriminating albendazole-resistant genotypes of H. contortus, providing an effective tool for anthelmintic selection and resistance surveillance in veterinary clinical practice.