title: Development of Tm-shift genotyping method for detection of cat-derived Giardia lamblia.
abstract: To develop Tm-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 Tm-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 Tmvalues 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 × 10-5and 4.88 × 10-5ng/μL samples of assemblage A and F standard plasmids. The Tm-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 Tm-shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the Tm-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.
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Weida
authors.initials: W
authors.lastname: Pan
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Yeqi
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Auwalu Yusuf
authors.initials: AY
authors.lastname: Abdullahi
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Mingwei
authors.initials: M
authors.lastname: Wang
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Xianli
authors.initials: X
authors.lastname: Shi
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Xingang
authors.initials: X
authors.lastname: Yu
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Yan
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China.
authors.forename: Jianxiong
authors.initials: J
authors.lastname: Hang
authors.affiliation: Guangdong Provincial Zoonosis Prevention and Control Key Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510542, China. gqli@scau.edu.cn.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Li
mesh_terms: D000818:Animals; D002371:Cat Diseases / Q000175:diagnosis* / Q000469:parasitology; D002415:Cats; D003598:Cytoskeletal Proteins / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016829:Giardia lamblia / Q000235:genetics*; D005873:Giardiasis / Q000175:diagnosis* / Q000469:parasitology; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D003598:Cytoskeletal Proteins; D017931:DNA Primers; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C038207:giardin protein, Giardia lamblia
keywords: Cat; Giardia lamblia; SNP; T m -shift genotyping; β-giardin gene