Alternative methods for yeast detection and identification
Although the detection methods described above provide accurate and reliable information on the presence of culturable (live) cells, these tests can be labour-intensive and require anywhere from 3-14 days to obtain results. This has prompted alternative techniques to be developed that are less laborious and provide results more rapidly, resulting in a more immediate, rather than retrospective, process analysis. Although there are many methods available for detection of beer-spoiling yeasts, the most widely employed are those that utilize polymerase chain reaction (PCR) technology. This technique permits the amplification of DNA fragments (corresponding to specific organisms) by several orders of magnitude over a few hours. Consequently, using PCR it is possible to amplify extremely small amounts of DNA to levels that can be used for detection and identification purposes; an approach that lends itself to forensic science, medical microbiology, food microbiology, as well as brewing quality assurance. The PCR procedure consists of repeated cycles of DNA denaturation, primer annealing, and extension by DNA polymerase, and relies on the selection of a DNA sequence that adequately differentiates the genotype of one organism from another. The amplified DNA is then visualized either by gel electrophoresis in conjunction with a DNA stain such as ethidium bromide (standard PCR), or by using primers in conjunction with a probe to provide a quantitative indication of PCR amplification through the production of fluorescence (quantitative real-time PCR, or qPCR). One basic example is to employ primers designed for detection of the STA1 gene responsible for diastatic activity in Saccharomyces strains (Yamauchi et al., 1998), although a range of DNA sequences that are unique to different species can be exploited.
It is relatively common for larger brewing companies to use commercially available PCR kits, as these provide a rapid and reliable means of both detecting and identifying beer-spoiling yeasts in a single reaction. Implementation of PCR technology has gained momentum since the first commercially available kits became available in the early 2000s. These have gained in popularity, partly because of the development of equivalent methodology for bacterial detection, which allows a range of brewing microbes to be investigated in parallel. However, the primary reason is that such methods are easy to perform on a routine basis and provide results that are often supported by data available on-line, allowing for increased confidence in the data obtained. Despite this, it should be recognized that such tools are not amenable to many brewery laboratories since the level of expertise required is relatively high and costs are often prohibitive. In addition, there are still issues with regard to inhibition of the PCR reaction by beer components, and the fact that PCR methods do not easily discriminate between live and dead cells. Finally, the level of sensitivity remains low (around 1 x 102 - 1 103 cells/ml), which often necessitate a period of ‘pre-enrichment' to ensure that false negatives are eliminated. As a result of these factors, many breweries continue to perform traditional testing as a back-up measure, indicating that there may still be an element of caution in relying solely on PCR. Despite this, due to the advances in next-generation sequencing, it is likely that DNA-based techniques will continue to develop. The capacity to identify beer-spoiling yeast species by mechanisms such as fluorescence in situ hybridization (FisH) (Xufre et al., 2006), or PCR analysis based on single nucleotide polymorphisms (SNPs) (Wilkening et al., 2013) or ribosomal DNA sequence divergence offer interesting opportunities for the future.