1. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR
- Author
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Cédric Longin, Hervé Alexandre, Michèle Guilloux-Benatier, Procédés Alimentaires et Microbiologiques ( PAM ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Vin Aliment Microbiologie et Stress ( VAlMiS ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Procédés Alimentaires et Microbiologiques [Dijon] ( PAM ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté ( UBFC ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté ( UBFC ), Procédés Alimentaires et Microbiologiques (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Vin Aliment Microbiologie et Stress (VAlMiS), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Procédés Alimentaires et Microbiologiques [Dijon] (PAM), and Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université Bourgogne Franche-Comté [COMUE] (UBFC)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement
- Subjects
0301 basic medicine ,Microbiology (medical) ,Polyvinylpolypyrrolidone ,030106 microbiology ,Population ,Food spoilage ,lcsh:QR1-502 ,Biology ,Microbiology ,lcsh:Microbiology ,Matrix (chemical analysis) ,03 medical and health sciences ,chemistry.chemical_compound ,[SDV.IDA]Life Sciences [q-bio]/Food engineering ,education ,Acetic acid bacteria ,DNA extraction ,Original Research ,Wine ,education.field_of_study ,Chromatography ,Red wine ,[ SDV.IDA ] Life Sciences [q-bio]/Food engineering ,biology.organism_classification ,3. Good health ,Microbiological internal control ,Real time PCR ,Real-time polymerase chain reaction ,chemistry ,Biochemistry - Abstract
International audience; Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.
- Published
- 2016