A real-time assemblage-specific PCR assay for the detection of Giardia duodenalis assemblages A, B and E in fecal samples.
Giardiosis is a common gastrointestinal infection caused by the flagellate Giardia duodenalis, and affects both humans and animals, worldwide. Animals are infected with both zoonotic and host-specific G. duodenalis assemblages, and their role in the transmission of the infection to humans has been a subject of intense research and debate. Conventional PCR assays are appropriate to determine G. duodenalis assemblages, but lack sensitivity for the detection of mixed infections. Previous surveys demonstrated the occurrence of mixed infections with G. duodenalis assemblage A and B in humans, and with assemblages A and E in cattle, but are likely to be underestimated. In this study, we designed a set of assemblage-specific primers by exploiting sequence variability in homologous genes from assemblages A, B and E. Primers were designed to amplify fragments of different size that generated different melting curves from each assemblage in real-time PCR (rt-PCR) experiments. The assay has been tested on a large panel of human and farm animal isolates, and shown to possess high specificity (no cross reactions observed) and sensitivity (detection limit close to 20 copies). Therefore, this assay can be useful to detect zoonotic and host-specific G. duodenalis assemblages in fecal samples from farm animals, particularly when a large number of samples is to be tested.
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
Lindy
L
Van Lith
Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Barbara
B
Šoba
Faculty of Health Science, Universidad de Magdalena, Colombia.
Vivian Villalba
VV
Vizcaino
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Staffan
S
Svard
Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and Environment (RIVM), Bilthoven, The Netherlands.
Hein
H
Sprong
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
Fabio
F
Tosini
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
Edoardo
E
Pozio
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy. Electronic address: simone.caccio@iss.it.
Simone M
SM
Cacciò
D000818:Animals; D000829:Animals, Domestic; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000469:parasitology; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D005243:Feces / Q000469:parasitology; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D005873:Giardiasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D058507:Host Specificity; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D013045:Species Specificity; D015047:Zoonoses
D017931:DNA Primers; D016054:DNA, Protozoan
Assemblage-specific assays; Farm animals; Giardia duodenalis; Molecular diagnosis; Real-time PCR; Zoonoses