Development of real-time PCR methods for the rapid detection of low concentrations of Gluconobacter and Gluconacetobacter species in an electrolyte replacement drink

Abstract

Aims: To develop a rapid real‐time polymerase chain reaction (PCR) method to detect Gluconobacter and Gluconacetobacter species in electrolyte replacement drinks. Methods and Results: Samples of electrolyte replacement drinks were artificially contaminated with Gluconobacter species and then filtered to collect cells. DNA was extracted from the filters and analysed by real‐time PCR on the ABI Prism 7000 system, using commercial detection kits for lactic and acetic acid bacteria. In addition, specific primers and Taqman® probe were designed and used for the detection of seven Gluconobacter and Gluconacetobacter species. All the assays tested demonstrated a linear range of quantification over four orders of magnitude, suggesting detection levels down to 1 CFU ml−1 in the original drink. Conclusions: A real‐time PCR method was developed to detect low concentrations of Gluconobacter and Gluconacetobacter sp. in an electrolyte replacement drink. Significance and Impact of the Study: Real‐time PCR methods allow a rapid, high throughput and automated procedure for the detection of food spoilage organisms. The real‐time PCR assay described is as sensitive as the conventional method that involves pre‐enrichment, enumeration on a selective agar (typically malt extract agar) and identification with a differential medium (typically Wallerstein nutrient agar). The real‐time PCR assay also provides a more rapid rate of detection, with results in less than 24 h following enrichment for Gluconobacter and Gluconacetobacter species.

Noninvasive Analysis of Microbiome Dynamics in the Fruit Fly Drosophila melanogaster

“…(19). To obtain comparisons of the relative abundance of each species, we corrected for primer efficiencies and determined the threshold cycle (C T ) values relative to those for total bacteria in each sample, i.e., we averaged the C T values between technical duplicates and then applied the following equation: ⌬C T ϭ C T(V2 primer set) Ϫ C T(sample bacteria-specific primer sets) .…”

Gluconobacter cerevisiae sp. nov., isolated from the brewery environment

“…The genus Gluconobacter belongs to the family Acetobacteraceae within the class Alphaproteobacteria and currently comprises 13 species with validly published names. Members of the genus Gluconobacter oxidize glucose to gluconic acid (De Ley & Frateur, 1970; Gammon et al , 2007) rather than ethanol to acetic acid, differentiating them from most acetic acid bacteria (AAB) (Andrés-Barrao et al , 2013; De Ley & Frateur, 1970). They are unable to oxidize acetate to carbon dioxide and water (Yamada & Yukphan, 2008).…”

Gram-negative spoilage bacteria in brewing

“…Rapid detection of AAB using real-time polymerase chain reaction (PCR) (Gammon et al, 2007;Torija, Mateo, Guillamón, & Mas, 2010), restriction fragment length polymorphism (Ruiz, Poblet, Mas, & Guillamon, 2000;Nanda et al, 2001), amplified fragment length polymorphism (Cleenwerck, de Wachter, Gonzalez, de Vuyst, & de Vos, 2009), denaturing gradient gel electrophoresis (Andorrà, Landi, Mas, Guillamón, & Esteve-Zarzoso, 2008;De Vero et al, 2006) and fluorescence in situ hybridisation (Frank et al, 1999;Franke-Whittle, O'Shea, Leonard, & Sly, 2005) are some of the examples of molecular detection methods. Various rapid detection techniques for AAB have also been described in the literature.…”