Differentiation of Species Belonging to <i>Saccharomyces</i> Sensu Stricto Using a Loop-Mediated Isothermal Amplification Method

Hayashi, Naoaki; Minato, Toshiko; Kanai, Keiko; Ikushima, Shigehito; Yoshida, Satoshi; Tada, Setsuzo; Taguchi, Hiroshi; Ogawa, Yutaka

doi:10.1094/asbcj-2009-0309-01

Cited by 4 publications

(5 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ultimately, a successfully amplified STA1 gene could be detected with a CRISPR-Cas12a reaction combined with two different visual detection methods (fluorescence and lateral flow). The STA1 gene could also be detected directly using only LAMP amplification and gel electrophoresis as in Figure 4, and this has also been previously demonstrated (Hayashi et al 2009), or as change in fluorescence or colour with DNA-binding dyes (Dao Thi et al 2020). However, isothermal methods tend to have a high rate of non-specific amplification, but CRISPR-Cas systems, on the other hand, have high specificity and sensitivity (Mahas et al 2021).…”

Section: Resultssupporting

confidence: 58%

“…Of the three compared pre-amplification methods, LAMP appeared to be the most promising in that it could clearly differentiate a STA1+ from a STA1- strain, and the reaction could be performed isothermally. PCR and LAMP primers for STA1 gene detection have been previously developed (Yamauchi et al 1998; Hayashi et al 2009), but the existing primers could not be used here as they do not produce amplicons containing the Cas12a protospacer sequence. Primer design to produce amplicons containing the Cas12a protospacer sequence proved challenging, which is evident from the poor specificity observed during the RPA amplifications and some of the PCR primers, and the failed amplification for the second set of LAMP primers.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Uotila

Krogerus

2022

Preprint

View full text Add to dashboard Cite

Diastatic Saccharomyces cerevisiae is a common contaminant in the brewing industry. Currently available detection methods are either time-consuming or require specialized equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from beer and yeast samples. More specifically, we aimed to develop a simple and rapid assay that requires minimal laboratory equipment or training, and ideally yields results as accurate as PCR-based methods. The developed assay consisted of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a-based detection and visualisation. We compared different pre-amplification and visualisation techniques, 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 only required a heat block, a pipette, and a centrifuge. The assay result was then visualised on a lateral flow strip. We used the developed assay to monitor an intentionally contaminated beer fermentation, and it was shown to yield results as accurate as PCR using previously published primers. Furthermore, the assay yielded results in approx. 75 minutes starting from a beer sample. The developed assay therefore offers reliable and rapid quality control for breweries of all sizes and can be performed without any expensive laboratory equipment. We believe the assay will be particularly useful for smaller breweries that don't already have well-equipped laboratories and are looking to implement better quality control.

show abstract

Section: Resultssupporting

confidence: 58%

Section: Resultsmentioning

confidence: 99%

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

Uotila

Krogerus

2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Ultimately, a successfully amplified STA1 gene could be detected with a CRISPR-Cas12a reaction combined with two different visual detection methods (fluorescence and lateral flow). The STA1 gene could also be detected directly using LAMP amplification and gel electrophoresis (Figure 3), as previously demonstrated (Hayashi et al 2009), or through a change in fluorescence or colour with DNA-binding dyes (Dao Thi et al 2020). However, because of the high amplification efficiency of LAMP, and products with multiple repeats, the technique is prone to non-specific amplification and false positives (Ho et al 2018;Bao et al 2020;Zhang et al 2021).…”

Section: D Bmentioning

confidence: 73%

“…-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%

“…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. Primer design to produce amplicons containing the Cas12a protospacer sequence proved challenging, evident from the poor specificity during the RPA amplifications and some of the PCR primers, and the failed amplification for the second set of LAMP primers.…”

Section: Figure 2(b)mentioning

confidence: 99%

See 1 more Smart Citation

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

Uotila

Krogerus

2023

J. Inst. Brew.

View full text Add to dashboard Cite

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.

show abstract

Current awareness on yeast

2009

Yeast

View full text Add to dashboard Cite

In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Reviews; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (5 weeks journals ‐ search completed 5th. Aug. 2009)

show abstract

Differentiation of Species Belonging to Saccharomyces Sensu Stricto Using a Loop-Mediated Isothermal Amplification Method

Cited by 4 publications

References 30 publications

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

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

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

Current awareness on yeast

Contact Info

Product

Resources

About