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Sensitive and rapid detection of Vibrio corallilyticus by loop‐mediated isothermal amplification targeted to the alpha subunit gene of RNA polymerase
Author(s) -
Liu G.F.,
Wang J.Y.,
Xu L.W.,
Ding X.,
Zhou S.N.
Publication year - 2010
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2010.02894.x
Subject(s) - loop mediated isothermal amplification , microbiology and biotechnology , polymerase chain reaction , biology , recombinase polymerase amplification , detection limit , gene , multiple displacement amplification , dna , chemistry , chromatography , genetics , dna extraction
Aims: A diagnostic protocol was developed for rapid detection of Vibrio corallilyticus by method of loop‐mediated isothermal amplification (LAMP). Methods and Results: For cloning and sequencing of rpo A gene of V. corallilyticus , a set of four LAMP primers were designed by targeting the rpo A gene. With Bst DNA polymerase, the reaction time and temperature were optimized for 70 min at 65°C, respectively. The amplification products were detected by electrophoresis. The detection limit of V. corallilyticus by LAMP was 3·6×10 3 CFU ml −1 (8 CFU per reaction), but PCR could detect up to 3·6×10 4 CFU ml −1 (72 CFU per reaction). The LAMP method was ninefold more sensitive than conventional PCR. The results also indicated that the LAMP reaction was highly specific to V. corallilyticus . Conclusions: The LAMP assay was a sensitive, specific and cost‐effective method for the rapid detection of V. corallilyticus . Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. corallilyticus infection. It can replace laborious biochemical tests for the identification of V. corallilyticus .