Study On Three Nucleic Acid Amplification Technology For Sensitive Detection Of Acetobacter Aceti In Yoghurt

Acetic acid bacteria are bacteria that have a unique ability to incompletelyoxidize various sugars and alcohol to organic acids.This ability has utilized forvinegar production, kombucha tea fermentation and cocoa fermentation in industricalproduction. But acetic acid bacteria can also spoil other foods and beverages, such asfruits, beer, wine and soft drinks. In resent years, studies show that acetic acid bacteriacan also spoil yogurt and metabolize the nutrition from yogurt, lead to vinegaryoff-flavors, pack swelling, turbidity and ropiness. But there are no standard test methods inthe domestic and overseas.Phenotypic methods and genotypical methods are the main methods for AABidentification. AAB have traditionally been identified by plating in selective solidculture media which can support growth of AAB. Different media are used forisolation and phenotypic methods based on physiological abilities were used foridentification. On the other hand, there are some limitations for plating such as timerequirement, labor intensive and inability to detect viable but noncultivable cells. Toovercome these disadvantages of culturing, new techniques have been developedusing molecular approaches, which overcome the deficiency of the phenotypicanalysis, with strong specificity and high sensitivity, they can detect the amount ofacetic acid bacteria in the actual conditions.This study selects Acetobacter aceti as the representative strain, conduct apreliminary study on testing method of the acetic acid bacteria pollution in yogurt. Weuse three genotypical technology involving LAMP, conventional PCR and Real-timePCR for Acetobacter aceti detection and compare the sensitivity of three kinds oftechnology, which include the following content:1. According to nucleic acid sequences of Acetobacter aceti (NBRC14818,GI39573569)16S-23S rRNA internal transcribed region sequence provided byGenbank, we finally selected a specific region and designed a set of LAMP specificprimers (https://primerexplorer.jp/lamp4.0.0/index.html). Then used the F3and B3as specific primers of the conventional PCR and SYBR Green Real-time PCR. Resultsindicate that these primers have satisfactory specificity by the specificity analysis. Thefollowing sequence of he primers were:F3：5’-CGGTTGGTGCGTATGTGG-3’；B3：5’-CCATGCACAGAAACCATCCA-3’；FIP:5’-ACGCTTCCGCCAAAATCCCTTGGGTTGGTCTGACCCATGA-3’；BIP:5’-AATGTGACGCGCTTGAATGAGCCCAAGGCAGAACCCCGATA-3’；2. Comparing the effect of three DNA extraction methods including heat-lysetreatment, DNA extraction kit method and CTAB extracting method, showed that theDNA extraction kit method was relatively stable, so we confirmed this method forDNA extraction of Acetobacter aceti.3. Optimized the reaction conditions in the LAMP reaction, and determined theoptimized system of25μL: MgCl26mmol/L,1.4mmol/L dNTPs, primers with aconcentration of0.8μmol/L inner primers of each FIP and BIP, and0.2μmol/L outerprimer of each F3and B3,1×Bst DNA polymerase reaction buffer,8U of the BstDNA polymerase large fragment,0.8mmol/L betaine,2μL isolated template DNA,and sterilized distilled water. The mixture was incubated at62℃for30minites,then heated to80℃for3minites to terminate the reaction.Then we improved thedetection methods after the LAMP reaction,and improved the visual effect by adding10000×0.1μL SYBR GreenⅠdye in the tube,which enhanced the visual inspectionsensitivity and reduced the detecting time.4. Achieved the effective quantitative detection of actobacter aceti by RT-PCRtechnology with SYBR Green dye. The standard curve between the number of copiesof mumerical and Ct value was: y=-3.3914x+41.919, with R2Value of0.9984.5. Comparison the sensitivity of LAMP technology, conventional PCR andSYBR Green Real-time PCR for detection in pure culture of Acetobacter aceti limitwere:3.63×10-1CFU/mL,3.63×101CFU/mL and3.63×101CFU/mL; the sensitivityfor detecting in artificial contamination Acetobacter aceti in yogurt were1.3× 102CFU/mL,1.3×104CFU/mL and1.3×101CFU/mL. Indicated that LAMP andSYBR Green RT-PCR technology showed the higher sensitivity.6. The Acetobacter aceti detecting results from deteriorated yogurt by theLAMP and SYBR Green RT-PCR technology concluded with the traditional culturemethod was100%. The LAMP and RT-PCR can detect Acetobacter aceticontamination in yogurt without enrichment. With high accuracy, they can apply onrapid detection of Acetobacter aceti contamination in yogurt.