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

• Published in: Parasitology research (1987) 2017-04, Vol.116 (4), p.1151-1157
• Main Authors: Pan, Weida, Fu, Yeqi, Abdullahi, Auwalu Yusuf, Wang, Mingwei, Shi, Xianli, Yang, Fang, Yu, Xingang, Yan, Xinxin, Zhang, Pan, Hang, Jianxiong, Li, Guoqing
• Format: Article
• Language: English
• Subjects: Animals; Cat Diseases - diagnosis; Cat Diseases - parasitology; Cats; Cytoskeletal Proteins - genetics; DNA Primers - genetics; DNA, Protozoan - genetics; Fish; Genotype; Genotypes; Genotyping; Genotyping Techniques - methods; Giardia lamblia - genetics; Giardiasis - diagnosis; Giardiasis - parasitology; Humans; Melting curve; Plasmids; Polymerase chain reaction; Polymorphism, Single Nucleotide - genetics; Primers; Protozoa; Protozoan Proteins - genetics; Real-Time Polymerase Chain Reaction; Single-nucleotide polymorphism
• ISSN: 0932-0113; 1432-1955
• DOI: 10.1007/s00436-017-5378-1

To develop T -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 -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 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 and 4.88 × 10  ng/μL samples of assemblage A and F standard plasmids. The T -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 -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T -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.