Rapid Identification of Fermentation Spoilage Microbes Using Molecular Beacons and a Two-Step Direct DNA Amplification Protocol

Shaw, Benjamin; Reekers, Corrine; Fenske, William; Fugate, Dylan; Brock, Martin; Fredericks, Jamie D.

doi:10.1080/03610470.2020.1712644

Cited by 1 publication

(2 citation statements)

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“…Probes are more specific than SYBR Green but tend to be more complicated to design and add significant cost per reaction. Molecular beacons are similar to Taqman probes, but have a unique sequence flanked by inverted repeats, which allow for a stem-loop structure to form and quench fluorescence when complimentary double-stranded DNA is not present ( Shaw et al, 2020 ). Probes and molecular beacons are used for multiplexed reactions, where multiple probes or beacons with different colors of fluorophore may be used to detect multiple targets during the same thermal cycler run ( Gilbride, 2014 ; Shaw et al, 2020 ).…”

Section: Methods Of Bsm Detectionmentioning

confidence: 99%

“…Much literature exists on BSM specific PCR methods, some of which utilize what they term “direct DNA extraction,” where DNA is amplified from its source without a significant extraction step. Often this includes simply isolating cells by centrifugation or membrane filtration and wash, then heating cells to cause cell lysis ( Juvonen and Haikara, 2009 ; Shaw et al, 2020 ). However in most publications found on BSM specific PCR methods samples were either sub-cultured, spiked, or enriched prior to DNA extraction potentially adding days to the time it takes to obtain PCR results ( Juvonen and Satokari, 1999 ; Haakensen et al, 2007 ; Juvonen et al, 2008 ; Juvonen and Haikara, 2009 ; Shaw et al, 2020 ; Kurniawan et al, 2021 ).…”

Section: Methods Of Bsm Detectionmentioning

confidence: 99%

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Methods for detection and identification of beer-spoilage microbes

Oldham

Held

2023

Front. Microbiol.

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It is critical that breweries of all sizes routinely monitor the microbiome of their process to limit financial losses due to microbial contamination. Contamination by beer-spoiling microbes (BSMs) at any point during the brewing process may lead to significant losses for breweries if gone undetected and allowed to spread. Testing and detection of BSMs must be routine and rapid, and because even small breweries need the capability of BSM detection and identification, the method also needs to be affordable. Lactic acid bacteria (LAB) are responsible for most spoilage incidents, many of which have been shown to enter the viable but nonculturable (VBNC) state under conditions present in beer such as cold or oxidative stress. These bacteria are invisible to traditional methods of detection using selective media. This article describes several methods of BSM detection and identification that may be useful in the majority of craft breweries. While there are several genomic methods that meet some or many qualifications of being useful in craft breweries, real-time quantitative polymerase chain reaction (qPCR) currently best meets the desired method characteristics and holds the most utility in this industry, specifically SYBR Green qPCR. qPCR is a targeted method of detection and identification of microbes that is affordable, rapid, specific, sensitive, quantitative, and reliable, and when paired with valid DNA extraction techniques can be used to detect BSMs, including those in the VBNC state.

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Section: Methods Of Bsm Detectionmentioning

confidence: 99%