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

Yeqi Fu; Yunqiu Liu; Asmaa M.I. Abuzeid; Yue Huang; Xue Zhou; Long He; Qi Zhao; Xiu Li; Jumei Liu; Rongkun Ran; Guoqing Li

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자료유형: 학술저널

저자정보: Yeqi Fu (South China Agricultural University) Yunqiu Liu (South China Agricultural University) Asmaa M.I. Abuzeid (South China Agricultural University) Yue Huang (South China Agricultural University) Xue Zhou (South China Agricultural University) Long He (South China Agricultural University) Qi Zhao (South China Agricultural University) Xiu Li (South China Agricultural University) Jumei Liu (South China Agricultural University) Rongkun Ran (South China Agricultural University) Guoqing Li (South China Agricultural University)

저널정보: 대한기생충학열대의학회 Parasites, Hosts and Diseases The Korean Journal of Parasitology Vol.57 No.1

발행연도: 2019.2

수록면: 9 - 15 (7page)

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Melting temperature shift (Tm-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 Tm-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 Tm-shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The Tm-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 Tm- 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 Tm-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 Tm-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.

#Anoylostoma ceylanicum #Anoylostoma tubaeforme #cat #T< #SUB> #m< #/SUB> #-shift #SNP #ITS1

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