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Establishment of a Tm-shi 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 대한기생충학ㆍ열대의학회 2019 The Korean Journal of Parasitology Vol.57 No.1
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 ceyl- anicum 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 distin- guish 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 concen- tration 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, sensi- tive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.
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Sequence Analysis of Mitochondrial Genome of Toxascaris leonina from a South China Tiger
Kangxin Li,Fang Yang,A. Y. Abdullahi,Meiran Song,Xianli Shi,Minwei Wang,Yeqi Fu,Weida Pan,Fang Shan,Wu Chen,Guoqing Li 대한기생충학열대의학회 2016 The Korean Journal of Parasitology Vol.54 No.6
Toxascaris leonina is a common parasitic nematode of wild mammals and has significant impacts on the protection of rare wild animals. To analyze population genetic characteristics of T. leonina from South China tiger, its mitochondrial (mt) genome was sequenced. Its complete circular mt genome was 14,277 bp in length, including 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. The nucleotide composition was biased toward A and T. The most common start codon and stop codon were TTG and TAG, and 4 genes ended with an incomplete stop codon. There were 13 intergenic regions ranging 1 to 10 bp in size. Phylogenetically, T. leonina from a South China tiger was close to canine T. leonina. This study reports for the first time a complete mt genome sequence of T. leonina from the South China tiger, and provides a scientific basis for studying the genetic diversity of nematodes between different hosts.
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