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Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

ABSTRACT: Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63? by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1?) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

SUBMITTER: Li J

PROVIDER: S-EPMC3662070 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

Li Jie J Wang Peiyuan P Zhang Aiguo A Zhang Ping P Alsarakibi Muhamd M Li Guoqing G

The Korean journal of parasitology 20130425 2

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10(-1) to 10(-5) ng/µl for LAMP and PCR ...[more]

PMID: 23710094

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Project description:BackgroundVibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus.ResultsThe assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 x 10(2) CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13-22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.ConclusionThe LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.

Dataset Information

Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

Publications

Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

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