Study On LAMP And SYBR GREENⅠReal Time PCR For Detection Of Gluconobacter In Yoghourt

Gluconbacter was one of the three most important species belonging to acetic acid bacteria and was non-pathogenic Gram-negative bacteria widely found in nature. Gluconobacter have great value in the food and drug industrial production, especially in the field of vitamin C, Cellulose and miglitol. Although Gluconobacter would bring considerable economic benefits for the industrial, Gluconobacter would bring enormous economic losses for Yogurt manufacturer. It had been reported that AAB can change the structure of probiotics, correspondingly resulting in yogurt rancidity, discoloration, bag swollen and off flavor. Therefore, it was significant to establish a timely and effective detection assay for Gluconobacter in the production process. Loop-mediated isothermal amplification(LAMP) and SYBR Green I real-time PCR technology were applied and compared to detect Gluconobacter quickly and accurately.The 16 S rRNA X73820 gene sequence with high specificity in GenBank was identified as the target sequence for primer design by using primer design software. Combined with the primer analysis software and experiments excellent primers were screened. The outer primers(F3 and B3) were used to qRT-PCR.LAMP reaction system: 8 mmol/L MgCl2,1.0 mmol/L dNTPs,1.4 μmol/L FIP and BIP,0.2 μmol/L F3 and B3,1.0 μL Bst DNA enzyme,2.5 μL Bst DNA polymerase reaction buffer(20 mmol/L Tris-HCl(pH8.8),10 mmol/L KCl,10 mmol/L(NH4)2SO4,2 mmol/L MgCl2,0.1% Triton X-100),2.0 μL DNA template, and sterile distilled water to make up 25μL system. The reaction was terminated 61 ℃ for 40 min, and at 80 ℃ for 5 min to stop. SYBR Green I chromogenic method was feasibility to detect LAMP reaction visual inspection after compared three methods(Centrifugal, agarose gel electrophoresis and SYBR Green I chromogenic).Both methods have excellent specificity and the results showed that 3 Gluconbacter strains were positive and other strains were negative. Ct value by SYBR Green I qRT-PCR detection for Gluconbacter was Y=-3.411X+36.034, with R2=0.9984. Sensitivity LAMP and qRT-PCR methods detected Gluconobacter in pure culture were 75 CFU/mL, the sensitivity in artificial contamination yogurt were 210 CFU/mL and 21 CFU/mL, both methods detected Gluconobacter successfully in simulated samples.Therefore, LAMP and qRT-PCR established in this study provides specificity and sensitive methods for Gluconobacter detection, they were valuable for application and exploitation in yoghurt industries.