title: Rapid and Sensitive Detection of Vibrio alginolyticus by Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene.
abstract: Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus-specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate-labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP-LFD technique for V. alginolyticus was 1.8 × 10(2) CFU/mL while that of PCR was 1.8 × 10(3) CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10(3) CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP-LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP-LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity.
authors.affiliation: a Department of Biology, Faculty of Science , Srinakharinwirot University , Bangkok , 10110 , Thailand.
authors.forename: Saranya
authors.initials: S
authors.lastname: Plaon
authors.forename: Siwaporn
authors.initials: S
authors.lastname: Longyant
authors.forename: Paisarn
authors.initials: P
authors.lastname: Sithigorngul
authors.forename: Parin
authors.initials: P
authors.lastname: Chaivisuthangkura
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015335:Molecular Probes / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D044149:Vibrio alginolyticus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015335:Molecular Probes