| Vibrio parahaemolyticus is an important human pathogen in China, which could cause acute astrointestinal illness, and cause sapraemia leading to death in some cases.At present, Vibrio parahaemolyticus has become one of most serious problems which have bad effect on public health in our country. The food poisoning caused by Vibrio parahaemolyticus is going up in recent years, especially in coast areas. It is necessary to develop reliable fast method of detection and supervise contamination status of sea products with Vibrio parahaemolyticus.At present, there are some methods to detect Vibrio parahaemolyticus, such as isolation and identification of Vibrio parahaemolyticus, immunological tests and molecular biological examination.The isolation and identification of Vibrio parahaemolyticus are time-consuming, labor intensive and lower sensitivity, the sensitivity and specificity of immunological methods are unsatisfactory, the PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens and are considered reliable alternatives for traditional bacteriological methods, however, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories.Loop-mediated isothermal amplification (LAMP), Notomi developed a novle nucleic acid amplification technology in 2000. LAMP assay was a specific, rapid and sensitive method of detecting viruses, fungi, bacteria and genetically modified food. Therefore, the establishment of LAMP technology for the detection of Vibrio parahaemolyticus was very important.A LAMP amplification-based detection of total Vibrio parahaemolyticus in shellfish is developed by targeting gyrB gene. which was used to design LAMP primers through primer explorer software(https://primerexplorer.jp/lamp3.0.0/index.html), The reaction conditions were optimized including temperature, time and Mg2+ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.6μmol/L concentration of each inner primer (FIP and BIP), a 0.2μmol/L concentration of each outer primer (F3 and B3), a 0.8μmol/L concentration of the loop primer(LF and LB), 0.6 mmol/L each deoxynucleoside triphosphate, 2 mmol/L Mg2+, 10×Bst DNA polymerase reaction buffer, 8U of the Bst DNA polymerase large fragment, 1μL isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 65℃for 40min and then heated to 80℃for 5min to terminate the reaction. We can obtain the results of detection when the white precipitate was observed by visual examination.Controls for method specificity were made with 13 Vibrio parahaemolyticus strains, 14 non-parahaemolyticus Vibrio and 19 non-Vibrio strains, the result indicates Vibrio parahaemolyticus was positive and other strains were negative. The effect of 6 methods of extracting DNA from Vibrio parahaemolyticus in shellfish was compared, the result indicates there are no strict requirements for the method of extracting DNA.This study explored LAMP technology rapidly detecting Vibrio parahaemolyticus; determined the sensitivity, artificial contamination detection limit and compared with the ordinary PCR detection method. The results showed the sensitivity of LAMP detection of Vibrio parahaemolyticus stains was 19CFU/mL, the detection limit of Vibrio parahaemolyticus in artificially contaminated samples were 87 CFU/g. Using the DNA extraction kit according to the manufacturer's instructions, from samples processing to report results, time-consuming was 2 h. In the control, the results of the sensitivity of PCR detection of Vibrio parahaemolyticus stains was 1900 CFU/mL, the PCR detection limits of Vibrio parahaemolyticus in shellfish artificially contaminated samples were 8700CFU/g. Using the same method of extracting DNA, from samples processing to report results, time-consuming was 4 h.The samples with bacteria amount of 1CFU/g can be detected after enrichment of 2 h with a detection cycle of 4 h. In this study, real detection was made. The result indicates: the sensitivity of the method is 100%, the specificity is 98.72%, and the coincidence rate is 98.84%.The result indicates LAMP has the advantage of simplicity, rapidity, specificity and cost-effectiveness. In our study, we had developed a technology platform for the rapid detection of Vibrio parahaemolyticus in shellfish. |
