Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces.
A nested PCR assay (TPILC-PCR) was developed to detect and distinguish between Giardia duodenalis assemblages A and B from human faeces by analysis of the triose phosphate isomerase gene (tpi). The assay comprised an initial multiplexed block-based amplification. This was followed by two separate real-time PCR assays specific for assemblages A and B using a LightCycler and SYBR Green I to identify PCR products by melting-point analysis. RFLP analysis was applied to distinguish G. duodenalis assemblage A groups I and II. The real-time nested PCR was evaluated using DNA extracted from purified giardial trophozoites, Cryptosporidium oocysts, whole faeces containing a range of potential pathogens (including G. duodenalis), faecal smears and bacterial suspensions. The assay was specific, sensitive, reproducible and rapid.
Amar
Corinne F L
CFL
Health Protection Agency, Food Safety Microbiology Laboratory, Division of Gastrointestinal Infections, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK 2Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
Dear
Paul H
PH
Health Protection Agency, Food Safety Microbiology Laboratory, Division of Gastrointestinal Infections, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK 2Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
McLauchlin
Jim
J
Health Protection Agency, Food Safety Microbiology Laboratory, Division of Gastrointestinal Infections, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK 2Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.
D000818:Animals; D005243:Feces / Q000469:parasitology*; D005838:Genotype; D005872:Giardia / Q000235:genetics* / Q000302:isolation & purification*; D005873:Giardiasis / Q000175:diagnosis; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity