Rapid Detection Of Vibrioparahaemolyticus By Loop-Mediated Isothermal Amplification Method And The Application Of Nanomaterials In The System

Vibrio parahaemolyticus (VP) is one of foodborne pathogens widely prevalent in the southeast region of China, associated with acute gastrointestinal symptoms such as diarrhoea and vomiting, sometime causing seriously epidemic problem. Loop-mediated Isothermal Amplification (LAMP) is a simple, rapid, sensitive method with specific molecular detection. A highly conservative region of pR72H gene of VP was used as the target template for 2 pairs of LAMP primers. The LAMP reaction was observed by the precipitation and fluorescence of amplified DNA. The validation by electrophoresis, restriction enzyme digestion and samples analysis, and specificity and sensitivity of the method were also analyzed. The resolution of the detection was at lfg/μL DNA molecules without cross-reaction with other experimental strains, which was much higher than normal PCR in sensitivity and specificity. By analyzing 53 aquatic seafood samples, all results were fully consistent with the method of PCR.Based on the LAMP detection method, we studied the function of the nanomaterials in LAMP reaction. In order to make the experiment becomes intuitive and fast, we set up the fluorescent quantitative detection method to observe and analyze the LAMP reaction. Through screening the nanomaterials of AuNPs, AgNPs, TiO2NPs, CNPs, CNT with LAMP method, we found that AuNPs accelerated initiation of the LAMP reaction, especially the AuNPs in diameter of 10 nm, but no significant effect on the final yield of the reaction. And AgNPs inhibited the LAMP reaction both in speed and yield, the inhibition is proportional to the concentration of AgNPs. In addition to AuNPs and AuNPs, we could not detect accurate results of TiO2NPs, CNPs, CNT which are water-insoluble with the method of the fluorescent quantitative and electrophoresis. Besides, we found that AuNPs could not be expanded to the reaction temperature range of the LAMP reaction.In this study, the mechanism of AuNPs affected the LAMP reaction was studied preliminary. Because of obvious red shift of the absorption peak of the AuNPs before and after the reaction, we believed that the combination of AuNPs with some of the components of the system was happened. In further research, we found that the ability of AuNPs to bind with the enzyme is strong, and the ability to bind with dNTPs and ssDNA is weak.