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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/ μ L and 5.03 × 10 −6  ng/ μ L. 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.

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