Identification of Beer-Spoilage Microorganisms Using the Loop-Mediated Isothermal Amplification Method

Tsuchiya, Youichi; Ogawa, Masahiro; Nakakita, Yasukazu; Nara, Yasunobu; Kaneda, Hirotaka; Watari, Junji; Minekawa, Harumi; Soejima, Takahiro

doi:10.1094/asbcj-2007-0227-01

Cited by 9 publications

(5 citation statements)

References 9 publications

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“…These methods do not require a thermal cycler like PCR, decreasing equipment costs, as reactions take place at a single temperature and can be performed in a heating block. LAMP requires 4–6 primers ( Tsuchiya et al, 2007 ) and CPA requires multiple primers including at least cross primer making CPA assay design more complex than some other methods ( Xu et al, 2012 ). Reactions can be multiplexed with RCA, though this requires designing padlock probes ( Soares et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, after the reaction takes place and DNA is amplified, white precipitates comprised of magnesium pyrophosphate are found in the reaction mixture. Thus, real-time monitoring of the LAMP reaction can be performed by real-time monitoring of turbidity of the reaction mixture, where the amount of turbidity directly corresponds to the amount of target DNA amplified ( Tsuchiya et al, 2007 ). All that is required for LAMP instrumentation is consistent heating to the desired assay temperature with equipment and detection equipment ( New England Biolabs, 2023 ).…”

Section: Methods Of Bsm Detectionmentioning

confidence: 99%

“…Primers were designed for four beer-spoilage bacteria ( Lactobacillus brevis , Lactobacillus lindneri , Pediococcus damnosous , and Pectinatus ) and used the method to identify isolated colonies grown on agar medium. They performed the multiplex reaction at 65°C, and determined the limit of detection of this assay to be six copies of target DNA at the start of the reaction ( Tsuchiya et al, 2007 ). Additionally, LAMP has been used to detect and identify Lactobacillus brevis, L. lindneri, L. backii, and Paracollinoides .…”

Section: Methods Of Bsm Detectionmentioning

confidence: 99%

See 2 more Smart Citations

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: Discussionmentioning

confidence: 99%

Section: Methods Of Bsm Detectionmentioning

confidence: 99%

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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“…Probably the most frequently used nucleic acid-based method is the polymerase chain reaction (PCR) 32,36,46,54,62,70,83,84 , which is also commercially available for breweries. PCR and other amplification techniques 100 have been applied in detection of both marker genes and ribosomal RNA (rRNA). Another commonly used technique is rRNA hybridization, which is employed in fluorescence in-situ hybridization (FISH) 94,95 and sandwich hybridization assay (SHA) 44 , both allowing detection within a couple of days after detection.…”

Section: Alternative Detection Methodsmentioning

confidence: 99%

Enrichment Cultivation of Beer-Spoiling Lactic Acid Bacteria

Taskila

Kronlöf²,

Ojamo

2011

Journal of the Institute of Brewing

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Various molecular methods have been developed for the rapid microbiological analysis of beer and brewing process samples. However, enrichment cultivation is still needed in order to reach the detection limits of the molecular assays, and it may directly contribute to the costs and accuracy of detection. The selection of proper enrichment cultivation conditions may be complex due to the wide variety of available media, and controversial reports of their performance. Therefore, this article aims to clarify this process by summarizing the main factors affecting the growth of lactic acid bacteria (LAB) in the enrichment cultures and reported media for this purpose.

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“…-Of the three pre-amplification methods, LAMP was the most promising in that it could clearly differentiate a STA1 ⁺ from a STA1strain, and the reaction could be performed isothermally. LAMPbased protocols for the detection of beer spoilage microorganisms have been reported previously (Tsuchiya et al 2007;Hayashi et al 2009). PCR and LAMP primers for STA1 gene detection have been developed (Yamauchi et al 1998;Hayashi et al 2009), but the primers could not be used here as they do not produce amplicons containing the Cas12a protospacer sequence.…”

Section: Figure 2(b)mentioning

confidence: 99%

A simple and rapid CRISPR-Cas12a based detection test for diastatic Saccharomyces cerevisiae

Uotila

Krogerus

2023

J. Inst. Brew.

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Diastatic Saccharomyces cerevisiae is a common contaminant in the brewing industry. Currently available detection methods are either time consuming or require specialised equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from samples of beer and yeast. More specifically, the aim was to develop a simple and rapid assay that requires minimal laboratory equipment or training, and yields results as accurate as PCR-based methods. The assay consists of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a based detection and visualisation. Different pre-amplification and visualisation techniques were compared, and the final assay involved a one-pot reaction where LAMP and Cas12a were consecutively used to pre-amplify and detect a fragment from the STA1 gene in a single tube. These reactions required a heat block, a pipette, and a centrifuge with the assay result visualised on a lateral flow strip. The assay was used to monitor an intentionally contaminated brewing fermentation and was shown to yield results as accurate as PCR with previously published primers. Furthermore, the assay yielded results in approximately 75 minutes. The developed assay offers reliable and rapid quality control for breweries of all sizes and can be performed without expensive laboratory equipment. It is suggested that the assay will be particularly useful for smaller breweries without well-equipped laboratories who are looking to implement better quality control.

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Identification of Beer-Spoilage Microorganisms Using the Loop-Mediated Isothermal Amplification Method

Cited by 9 publications

References 9 publications

Methods for detection and identification of beer-spoilage microbes

Methods for detection and identification of beer-spoilage microbes

Enrichment Cultivation of Beer-Spoiling Lactic Acid Bacteria

A simple and rapid CRISPR-Cas12a based detection test for diastatic Saccharomyces cerevisiae

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