Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR

Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable techniques such as real-time PCR (rt-PCR) and nested PCR for enumerating and detecting the presence of this bacterial group without plating. Primers were designed on the basis of the available 16S rRNA gene sequences and tested successfully with reference acetic acid bacteria strains. The usefulness of rt-PCR was demonstrated by comparing the results with traditional techniques (colony and microscope counting). The results were similar with all the techniques. Optimized rt-PCR enabled numbers between 10(7) and 10(1) cells mL(-1) to be enumerated, while nested PCR detected less than 10 cells mL(-1). Although this latter technique cannot be used for enumeration, it has several advantages in routine laboratory analysis.