T_m-Shift Detection of Dog-Derived Ancylostoma ceylanicum and A. caninum

To develop a T_m-shift method for detection of dog-derived Ancylostoma ceylanicum and A. caninum, three sets of primers were designed based on three SNPs (ITS71, ITS197, and ITS296) of their internal transcribed spacer 1 (ITS1) sequences. The detection effect of the T_m-shift was assessed through the stability, sensitivity, accuracy test, and clinical detection. The results showed that these three sets of primers could distinguish accurately between A. ceylanicum and A. caninum. The coefficient of variation in their T_m values on the three SNPs was 0.09% and 0.15% (ITS71), 0.18% and 0.14% (ITS197), and 0.13% and 0.07% (ITS296), respectively. The lowest detectable concentration of standard plasmids for A. ceylanicum and A. caninum was 5.33 × 10^-6 ng/μL and 5.03 × 10^-6 ng/μL. The T_m-shift results of ten DNA samples from the dog-derived hookworms were consistent with their known species. In the clinical detection of 50 fecal samples from stray dogs, the positive rate of hookworm detected by T_m-shift (42%) was significantly higher than that by microscopic examination (34%), and the former can identify the Ancylostoma species. It is concluded that the T_m-shift method is rapid, specific, sensitive, and suitable for the clinical detection and zoonotic risk assessment of the dog-derived hookworm.