Glutamate dehydrogenase and triose-phosphate-isomerase coding genes for detection and genetic characterization of Giardia lamblia in human feces by PCR and PCR-RFLP
چکیده
Aim: The purpose of this study was comparison of the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes for detection and genetic characterization of Giardia lamblia (G. lamblia) in human stool by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP). Our assay was specific and distinguished between G. lamblia assemblages A and B. Materials and methods: Among the 325 stool samples obtained from patients with acute gastroenteritis hospitalized in the Pediatric Hospital and infected humans referred to the Tabriz Reference Laboratory (TRL), 34 Giardia-positive stool samples were identified by conventional techniques. Two assays PCR and PCR-RFLP targeting the tpi and gdh genes were developed to detect and genetically characterize G. lamblia isolates in human stool. Results: The tpi gene was amplified from 31(91.1%) samples by A-PCR and B-PCR assays. Of these samples 13 (41.9%) contained assemblage B, 17 (54.8%) contained assemblage A, 1 (3.2%) contained a mixture of assemblage A and assemblage B, and 3 (8.8%) samples were negative. Only 18 (52.9%) samples were positive when the gdh gene was targeted by GDH-PCR. RFLP analysis of the gdh gene classified 6 samples (33.33%) in assemblage A subgenotype II, 8 (44.44%) in assemblage B group III, and 4 (22.22%) in assemblage B group IV. Of these, 6 (33.33%) samples contained assemblage A and 12 (66.66%) samples contained assemblage B. Conclusion: These study results reveal that the PCR technique is sensitive, simple, and specific for Giardia detection in fecal samples. In addition, the results demonstrate that the tpi gene is well adapted for G. lamblia genotyping.