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Rapid and sensitive detection of V ibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene
Author(s) -
Thongkao Kanittada,
Longyant Siwaporn,
Silprasit Kun,
Sithigorngul Paisarn,
Chaivisuthangkura Parin
Publication year - 2015
Publication title -
aquaculture research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 89
eISSN - 1365-2109
pISSN - 1355-557X
DOI - 10.1111/are.12266
Subject(s) - vibrio harveyi , biology , loop mediated isothermal amplification , dipstick , amplicon , microbiology and biotechnology , vibrio , polymerase chain reaction , detection limit , bioluminescence , bacteria , gene , chromatography , biochemistry , chemistry , dna , genetics , urine
Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification ( LAMP ) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate ( FITC )‐labelled probe and lateral flow dipstick ( LFD ) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP ‐ LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐ harveyi Vibrio isolates, and 34 non‐ Vibrio bacterial isolates. The sensitivity of LAMP ‐ LFD for V. harveyi detection in pure culture was 1.1 × 10 2 CFU mL −1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10 3 CFU g −1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP ‐ LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii .