Quantitative loop-mediated isothermal amplification method for the detection of Vibrio nigripulchritudo in shrimp

• Published in: Fisheries science Vol. 77; no. 1; pp. 129 - 134
• Main Authors: Fall, Jean, Chakraborty, Gunimala, Kono, Tomoya, Maeda, Minoru, Suzuki, Yoshihiro, Itami, Toshiaki, Sakai, Masahiro
• Format: Journal Article
• Language: English
• Published: Japan Springer Japan 01.01.2011; Springer Nature B.V
• Subjects: Aquaculture; Biomedical and Life Sciences; Correlation coefficient; Deoxyribonucleic acid; Disease; DNA; DNA polymerase; Fish; Fish & Wildlife Biology & Management; Fishery sciences; Food Science; Freshwater & Marine Ecology; Life Sciences; Marine; Mortality; Original Article; Pathogens; Studies; Turbidity; Vibrio; Vibrio nigripulchritudo; Quantitative loop-mediated isothermal amplification; DNA; Intergenic spacer region
• ISSN: 0919-9268; 1444-2906
• DOI: 10.1007/s12562-010-0309-4

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10 2 copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo , whereas other Vibrio and non- Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time ( T t ) versus the log of bacterial number. A high correlation coefficient ( R 2  = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.