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Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens.

ABSTRACT: AIMS:?To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. METHODS AND RESULTS:?LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID?? ml?¹, whereas the detection limit of the PCR was 1000 TCID?? ml?¹. CONCLUSIONS:?The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. SIGNIFICANCE AND IMPACT OF THE STUDY:?The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.

SUBMITTER: Mukhopadhyay HK

PROVIDER: S-EPMC7197762 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens.

Mukhopadhyay H K HK Amsaveni S S Matta S L SL Antony P X PX Thanislass J J Pillai R M RM

Letters in applied microbiology 20120724 3

<h4>Aims</h4> To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation.<h4>Methods and results</h4> LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling ...[more]

PMID: 22748120

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Project description:BACKGROUND: Vibrio 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. RESULTS: The 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. CONCLUSION: The 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

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens.

Publications

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens.

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