Development of T m -shift genotyping method for detection of cat-derived Giardia lamblia

• DOI: 10.1007/s00436-017-5378-1
• Publication Date: 2017-02-14T23:15:48Z
• Authors: Weida Pan; Yeqi Fu; Auwalu Yusuf Abdu...; Mingwei Wang; Xianli Shi; Fang Yang; Xingang Yu; Xinxin Yan; Pan Zhang; Jianxiong Hang; Guoqing Li
• Keywords: Giardiasis; 0303 health sciences; Genotype; Genotyping Techniques; Protozoan Proteins; DNA, Protozoan; Cat Diseases; Real-Time Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Cytoskeletal Proteins; 03 medical and health sciences; Cats; Animals; Humans; Giardia lamblia; DNA Primers

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.