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      Recovery of DNA of Giardia intestinalis cysts from surface water concentrates measured with PCR and real time PCR Translated title: Amplification par PCR et PCR en temps réel d’ADN de kystes de Giardia intestinalis concentrés en eau de surface

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          The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max ® equipment (1623 Method). Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi-nested PCR was 15 and 20 ng/μl, whereas for real time PCR 5 ng/μl.

          Translated abstract

          La faible concentration des kystes de Giardia intestinalis dans les prélèvements d’eau de surface, ainsi que la présence d’inhibiteurs de la PCR constituent un handicap pour la détection du parasite. Nous avons effectué une série d’essais afin de tester la sensibilité de la PCR semi-nichée et la TaqMan PCR en temps réel dans la détection de l’ADN extrait de kystes de G. intestinalis. Ces kystes proviennent d’échantillons d’eau distillée et d’eau d’environnement, filtrés avec le Filta-Max ® (1623 Method). Les inhibiteurs ont été éliminés par la BSA utilisée à différentes concentrations. Des pertes de kystes ont été constatées lors des opérations de filtration et de concentration. L’addition de BSA augmente la sensibilité de la réaction. La concentration optimale de BSA était de 15 et 20 ng/μl pour la PCR semi-nichée et de 5 ng/μl pour la PCR en temps réel.

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          Most cited references 9

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          The zoonotic significance and molecular epidemiology of Giardia and giardiasis.

           R Thompson (2004)
          The taxonomy and molecular epidemiology of Giardia and Giardia infections are reviewed in the context of zoonotic and waterborne transmission. Evidence to support the zoonotic transmission of Giardia is very strong, but how frequent such transmission occurs and under what circumstances, have yet to be determined. Zoonotic origin for waterborne outbreaks of Giardia infection appears to be uncommon. Similarly, livestock are unlikely to be an important source of infection in humans. The greatest risk of zoonotic transmission appears to be from companion animals such as dogs and cats, although further studies are required in different endemic foci in order to determine the frequency of such transmission.
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            Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples.

            The flagellate parasite Giardia duodenalis is a major cause of diarrhoea in humans and in animals worldwide. Molecular techniques are particularly useful for studying the taxonomy, the population structure, the zoonotic potential of animal isolates, and the correlation between the genetic variability of the parasite and the range of clinical symptoms observed in humans. In this work, a new PCR assay that targets the beta-giardin gene was tested on 21 Giardia duodenalis reference strains representing Assemblages A, B and E, which are associated with infections of humans and other mammals. The assay was then applied to 30 faecal samples collected from Italian persons. The sequence analysis of 31 PCR products from both reference strains and clinical samples showed that each Assemblage is clearly distinct from the others on the basis of specific substitutions; the sequence diversity was approximately 5%, and all substitutions occurred at the third codon positions of the gene. The analysis of the intra-Assemblage variability allowed for the identification of three genotypes within Assemblage A, and of four genotypes within Assemblage B. Interestingly, two genotypes were identified only in the clinical samples and not in reference strains. Finally, a simple PCR-restriction fragment length polymorphism method was developed for the rapid discrimination of Assemblages and applied for the direct genetic analysis of cysts present in human faecal samples.
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              Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp.

              Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. are not only three of the most important and common diarrhea-causing parasitic protozoa, but they often have similar clinical presentations. Microscopic diagnosis of these parasites is neither sensitive nor specific. Recently, more specific and sensitive alternative molecular methods (polymerase chain reaction [PCR] and antigen detection tests) have been introduced for all three of these parasitic infections. The use of these molecular diagnostic tests in routine diagnostic laboratories is still limited. In this study, we developed a multiplex real-time PCR assay for the simultaneous detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. in one reaction using species-specific probes. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. The reagents used in this multiplex PCR assay can also be used for detection of these parasites individually. The use of this real-time PCR multiplex assay in developing countries at present will have limited scope for routine diagnosis because the cost will be high for a single test, although in the developed world, the test could see immediate application.

                Author and article information

                EDP Sciences
                November 2011
                15 November 2011
                : 18
                : 4 ( publisher-idID: parasite/2011/04 )
                : 341-343
                [1 ] Department of Genetics, University of Szczecin ul. Felczaka 3c 71-412 Szczecin Poland
                Author notes
                [* ]Correspondence: Bogumila Skotarczak. E-mail: boskot@ 123456univ.szczecin.pl
                parasite2011184p341 10.1051/parasite/2011184341
                © PRINCEPS Editions, Paris, 2011

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 1, Tables: 0, Equations: 0, References: 10, Pages: 3
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