Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia
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To develop T m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5′-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10−5 and 4.88 × 10−5 ng/μL samples of assemblage A and F standard plasmids. The T m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.
KeywordsGiardia lamblia Cat Tm-shift genotyping SNP β-giardin gene
This work was supported by grant from National Natural Science Foundation of China (grant nos. 31272551, 31672541). The authors would like to thank Prof. Zhaorong Lun from Southern China Research Center of Parasitic Biology, Sun Yat-sen University, China for offering G. lamblia trophozoites assemblages A.
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Conflict of interest
The authors declare that they have no conflict of interest.
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