Study On A Loop-Mediated Isothermal Amplification (Lamp) For The Fast Detection Of Vibrio Splendidus

In recent years, Vibrio splendidus is one of the main pathogenic bacteria in cultured sea cucumber. It can trigger skin ulcerative syndrome in cultured sea cucumber, which causing massive mortality and economic losses. In addition, Vibrio splendidus can also trigger many diseases in a variety of sea animals such as flounder, turbot, trout, Atlantic salmon, sea bass and others. At present, methods to detect Vibrio splendidus mainly include:generic detection of bacterial culture (spread plate method, identification of physiological and biochemical indexes); immunological tests based on antigen-antibody reaction (slide agglutination, double antigen sandwich ELISA, Dot-ELISA, indirect fluorescent rapid detection) and molecular biological examination (PCR method, oligonucleotide probe detection technology). However, the applications of these methods are still not very common at the grassroots level because of their complicated operation, expensive equipment, the sensitivity and specificity not ideal and time-consuming.In2000, Notomi proposed a novel nucleic acid amplification method: Loop-mediated isothermal amplification (LAMP). This method employs a DNA polymerase with strand displacement activity and a set of specially designed primers, which has many advantageous. LAMP method has been used in clinical diagnosis of infectious disease and research on nucleic acid etc. Therefore, applying LAMP method to the detection of Vibrio splendidus has a good foundation for scientific research.In this paper, we used Virulence Factors of Pathogenic Bacteria (VFDB) to determine the potential virulence related genes of Vibrio splendidus. We select the flagellar genes (flgF) that wildly existed in Vibrio bacteria as research object, and use it to design LAMP primers through primer explorer software (http://primerexplorer.jp/elamp4.0.0/index.html). A LAMP amplification-based detection of Vibrio splendidus was developed by target flgF gene. The primers specificity test was developed and the reaction conditions were optimized including reaction time, dNTP concentrations, Betaine concentrations and Mg2+concentrations. In addition, the sensitive detection of Vibrio splendidus was conducted and compared with the ordinary PCR detection method. The results are as follows:1. The primers used for the rapid detection of Vibrio splendidus in LAMP:F3:5’-GGATCGCGCACTGTTTCT-3’B3:5’-TGTGCGAAATTATGACCCGG-3’FIP:5’-ACCCGTTGTACTTACGTTGGCAGCCATGAGTGGCGCTAAG-3’BIP:5’-AGCACAAGCACGTTCAATGCAACCGTCATGCTGAAAACACGA-3’;2. The optimized reaction conditions are as follows:the concentration of out primer F3and B3,0.2μM; inner primer FIP and BIP,1.6μM; dNTP,1.0mM; Betaine,0.1M; Mg2+,5.0mM;8U of the Bst DNA polymerase large fragment; DNA template,1μL;10×Bst DNA polymerase reaction buffer,2.5μL; sterilized double-distilled water to25μL. The mixture was incubated at65℃for30～45min.3. The primers specificity test was developed with4Vibrio splendidus strains and12other strains. The result showed that only the4Vibrio splendidus were positive, indicating that the specificity of the primers are good.4. The results showed that the detection limit of LAMP detection of Vibrio splendidus genomic DNA was38fg/μL,100times higher than the ordinary PCR detection method. The detection limit of LAMP detection of Vibrio splendidus strains was1.3×103cfu/mL,100times higher than the ordinary PCR detection method. This indicated that the LAMP amplification-based detection of Vibrio splendidus has a high sensitivity.5. Results Interpretation:Fluorescent dye Calcein-Mn2+can realize closed-tube quick interpretation of the results.In summary, the LAMP amplification-based detection of Vibrio splendidus has a good application value with high specificity and sensitivity, low-cost device, simple operation, short time-consuming.