|
KMID : 0425120190570010009
|
|
Parasites, Hosts and Diseases
2019 Volume.57 No. 1 p.9 ~ p.15
|
|
|
Establishment of a Tm-shift Method for Detection of Cat-Derived Hookworms
|
|
Fu Yeqi
Liu Yunqiu
Abuzeid Asmaa M. I.
Huang Yue
Zhou Xue
He Long
Zhao Qi
Li Xiu
Liu Jumei
Ran Rongkun
Li Guoqing
|
|
|
Abstract
|
|
|
|
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.
|
|
|
KEYWORD
|
|
|
Anoylostoma ceylanicum, Anoylostoma tubaeforme, cat, Tm-shift, SNP, ITS1
|
|
|
FullTexts / Linksout information
|
|
 
|
|
|
Listed journal information
|
|
    
|
|