Comparative analysis of two methods for detection the DNA of acetic acid bacteria by real‐time PCR

Abstract

Acetic acid bacteria (AAB) are very difficult to correctly identify at species levels based only on biochemical and physiological characteristics. For their proper identification, molecular analysis of the strains in comparison with reference species is recommended. In recent years, a variety of methods based on molecular techniques of DNA extraction and identification by polymerase chain reaction (PCR) have been used for identification of the genera, species and strains of AAB. The study used a method Real-Time PCR allows the qualitative detection of the members of the acetic acid bacteria group including the well-known Acetobacter, Gluconacetobacter and Gluconobacter species. The goal of this study was comparing two methods of DNA isolation from vinegar for further analysis by real-time PCR for detection of acetic acid bacteria (AAB). In this work, the Detection Kit B Acetics Screening from PIKA was used. The DNA was isolated using the same kit as recommended by manufacturer, and using DNAzol Reagent. For the study, acetic acid bacteria were isolated from unpasteurized white wine and apple vinegar in RAE medium at 30 ° C, 48-72 h. As a result, we could identify the acetic acid bacteria from white wine and apple vinegar in the DNA samples isolated by two different methods. DNA was isolated from in equal quantities of the culture using DNAzole, amplification was successful and no inhibition of PCR was observed. The Ct values for this method were slightly lower for both samples compared to the values obtained for purified DNA using the kit buffer. The results of Ct obtained show that both methods of DNA extraction from acetic acid bacteria yielded amplifiable DNA, though in case of DNAzol the yield was slightly inferior compared to the genuine kit protocol.