Primer set for detection of saccharomyces yeast
A primer set, yeast technology, applied in the field of PCR primer set, detection and quantification of yeast of the genus Saccharomyces, LAMP primer set
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0217] Example 1: Detection of yeasts of the genus Saccharomyces
[0218] (a) Genomic DNA extraction method
[0219] The cells cultured on the agar plate medium were scraped from the medium, and then they were suspended in sterilized distilled water. The suspension was centrifuged (15,000 rpm, 5 minutes), and the supernatant was discarded. Sterilized distilled water was added to the reprecipitated cells, followed by suspending the mixed solution and centrifuging. The supernatant was discarded, and then 100 µl of a solution of PrepMan Ultra (manufactured by Applied Biosystems) was added to the obtained cells. The mixture was heated at 95°C for 10 minutes. Thereafter, the resultant was centrifuged at 15,000 rpm for 1 minute, and the supernatant was used as a genomic DNA solution. Separately, 100 μl of 0.1 N NaOH solution was added to the washed cells, and then the mixture was heated at 95° C. for 10 minutes. Thereafter, the resultant was neutralized with 1M Tris buffer (pH ...
Embodiment 2
[0279] Embodiment 2: the detection limit of LAMP method
[0280] To analyze the amplification efficacy of the LAMP method, cells of bottom-fermenting yeast (BFY70, Saccharomyces pastor), Saccharomyces cerevisiae NBRC10217 and Saccharomyces bayanus NBRC1948 that had been cultured on agar plate medium were diluted with sterile water in a stepwise manner , and then, DNA was extracted by the aforementioned method. The LAMP method was performed on the extracted DNA. As a result, in the case of the LAMP method, even a small number of cells such as 10 2 to 10 3 Amplification of cfu levels.
[0281] In addition, when using genomic DNA extracted from the diluted cell solution in each dilution step, the time when the turbidity exceeds 0.1 in the LAMP reaction was defined as the detection time, based on the logarithm of the detection time of each primer and the colony Form number form chart. As a result of the exponential approximation, an approximation curve with a high correlati...
Embodiment 3
[0288] Example 3: Detection of cells in wine and beer
[0289] In general, in the production of wine, a large number of yeasts of the genus Saccharomyces are used as wine yeast and added to fruit juice for fermentation. The number of yeast cells from externally contaminated juice was significantly smaller than that of Saccharomyces spp. Therefore, a large amount of Saccharomyces cerevisiae was suspended in wine, and thereafter, a dilute solution of cells of Saccharomyces bayanus NBRC1948 as prepared by diluting wine was mixed therewith, followed by collecting and washing the cells. Thereafter, the cells are collected and DNA is extracted from them. Using SBR2LB1 as a primer set, the LAMP method was performed, thus analyzing the possibility of detecting Saccharomyces japonicus. As a result, it was found that regardless of the inhibition of the reaction by wine and the presence of Saccharomyces cerevisiae, Saccharomyces cerevisiae could be detected at almost the same detecti...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More