Rapid Detection Of Acetic Acid Bacteria Group In Wine By RT-FQPCR And MALDI-TOF MS

A real-time fluorescence quantitative polymerase chain reaction(RT-FQPCR)and a matrix-assisted laser desorption ionization time-of-flight mass spectrometr(MADLI-TOF MS)were established to quickly and sensitively detect acetic acid bacteria in wine.Combining two methods with complementary advantages to detect acetic acid bacteria in wine and planning to construct a SARAMIS database of seven strains of acetic acid bacteria that often appeared in wine.In the RT-FQPCR procedure,improved the extraction of deoxyribonucleic acid(DNA)in wine,referenced universal primers of acetic acid bacteria,optimized the conditions of RT-FQPCR.Meanwhile,used seven kinds of acetic acid bacteria to verify this method versatility,and specificity;In the MALDI-TOF MS procedure,seven strains of acetic acid bacteria were taken as the object,different plates、temperatures、time、and stirring speed on the different MALDI-TOF MS spectra were analyzed by comparative evaluation of different sample processing methods.Added the map of seven standard strains of acetic acid bacteria to the SARAMIS database;In the last chapter added seven strains in ten kinds of commercially available wines to verify the sensitivity of the two methods.(1)all the seven bacteria strains had obvious amplification curves;the non-target bacteria showed no amplification under this condition but higher specificity.The sensitiviity showed that G.oxidans:7 CFU/m L、A.aceti:1 CFU/m L、G.japonicus:3CFU/m L A Pasteurii:4 CFU/m L、A.Tropicalis:2 CFU/m L、G.cerinus:7 CFU/m L、K.Saccharivorans:1 CFU/m L.(2)The formic acid extraction method had many characteristic peaks,smooth baseline,high signal-to-noise ratio,and the quality of the map was better than the direct smear method;2 strains of acetic acid bacteria(G.cerinus,G.oxidans)were cultured with the common plate at 34℃for 72 h and 100 r/min could get the largest characteristic peak and the significant test results;Both A.Tropicalis,and K.Saccharivorans were cultured with GY plate at 30℃for 84 h and 100 r/min could get the largest characteristic peak and the significant test results.The A.Pasteurii was cultured with GY plate at 30℃for72 h and 100 r/min could get the largest characteristic peak and the significant test results.Both A.aceti and G.japonicus were cultured with common plate at34℃and 30℃for 75r/min and 100 r/min in 72 h could get the largest characteristic peak and the significant test results.The detectable rate of seven acetic acid bacteria strains in the library could be reach 99.90%.(3)7 strains of acetic acid bacteria can be detected by RT-FQPCR method,obviously it is reduced with the sensitivity of the strain,7 strains of acetic acid bacteria are detected in the MALDI-TOF MS instrument The limit is 10~3 CFU/m L,and the identification score can reach more than 80%.The above two methods provide a technical method for the detection of acetic acid bacteria in wine in the future,and a suitable detection method can be selected for detection according to actual needs.