Rapid and Sensitive Detection of Vibrio natriegens in Portunus trituberculatus with the Loop-Mediated Isothermal Amplification Test

Abstract

In order to detect Vibrio natriegens in Portunus trituberculatus, a loop-mediated isothermal amplification (LAMP) method was developed and evaluated. In this study LAMP primers were designed to correspond to the gyrB gene sequences. With Bst DNA polymerase, the LAMP assay was completed within 100 min at 65 C in a water bath. Amplification products were observed directly with agar gel or with the naked eye after the addition of SYBR Green I. The sensitivity of the LAMP assay for the detection of V. natriegens is about 1.32×10-2 fg/mL DNA template, whereas using duplex PCR the detection of V. natriegens was possible up to 13.2 pg/mL DNA template. There were no cross-reactions with other Vibrio strains indicating a high specificity of the LAMP. The novel LAMP assay in this study can be used as a valuable, rapid, and sensitive detection tool for the detection of V. natriegens both in the laboratory and for use in commercial aquaculture. The IJA appears exclusively as a peer-reviewed on-line open-access journal at http://www.siamb.org.il. To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden.