Establishment of a Tm-shift Method for Detection of Cat-Derived Hookworms

Abstract

Melting temperature shift ( T m -shift) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a T m -shift method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5′ end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of T m -shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The T m -shift method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme . The coefficient of variation (CV) of T m -values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was 5.22×10 −6 and 5.28×10 −6 ng/μl samples of AceP and AtuP, respectively. The accuracy of T m -shift method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the T m -shift detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.

Prevalence of enteric helminths and protozoa and identification of hookworm, threadworm and giardia spp. In cats in Bangkok and vicinity, Thailand

“…This study is the first report of A. tubaeforme in Thailand. A. tubaeforme is widely distributed including Australia, Brazil, Italy, Qatar, Spain, the United States of America and China (Fu et al, 2019b). Clinical signs of A. tubaeforme infection depends on the dose of infection.…”

“…This study also firstly reported A. tubaeforme in Thailand. A. tubaeforme is widely distributed including Australia, Brazil, Italy, Qatar, Spain, the United States of America and China (Fu et al, 2019b). Heavy A. tubaeforme infection results in decreasing pack cell volume and hemoglobin rapidly causing anemia suggesting that the hookworm positive cats should not be neglected.…”