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- Title
Rapid and sensitive detection of Vibrio harveyi by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene.
- Authors
Thongkao, Kanittada; Longyant, Siwaporn; Silprasit, Kun; Sithigorngul, Paisarn; Chaivisuthangkura, Parin
- Abstract
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 × 102 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 × 103 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.
- Subjects
VIBRIOSIS in fishes; FISH genetics; VIBRIO harveyi; FISH microbiology; GENE targeting; GENE amplification; AQUACULTURE
- Publication
Aquaculture Research, 2015, Vol 46, Issue 5, p1122
- ISSN
1355-557X
- Publication type
Article
- DOI
10.1111/are.12266