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rDNA and RNA detection

PCR and qPCR

Many molecular techniques that seek to profile the microbes within a community specifically target ribosomal genes or rDNA (i.e. 16S rDNA, 23S rDNA, rDNA inter-space regions) given the ubiquitous presence of rRNA (i.e. in both viable and non-viable cells) and the conserved nature of these sequences, enabling the ability to distinguish between species and isolates (Ben-Amor et al.,

2007). As brewers are often solely interested in the presence of viable cells, other genes that increase the discriminatory power of these nucleic acid techniques, such as elongation-factor genes or other hop tolerance genes can also be targeted ( Juvonen et al., 2010).

The polymerase chain reaction (PCR) and various adaptations thereof are arguably the most frequently used means of performing targeted- interrogations of the 16S rDNA and target genes of interest (i.e. hop tolerance genes) for BSR LAB (Haakensen et al., 2007; Pittet et al., 2011; Suzuki et al., 2004a,b). Perhaps the most important variation of traditional end-point PCR for beer spoilage

LAB detection is that of the multiplex PCR assay, which is used to interrogate multiple targets at one time. Distinct primers targeting separate genes or regions of interest have been used within both the brewing and wine industry to profile hop tolerance genes and/or to rapidly identify LAB genera and species (Haakensen et al., 2008; Petri et al., 2013; Pfannebecker and Frohlich, 2008). These tests give same-day results, require relatively low expertise to run, and have sensitive detection limits, thus presenting an attractive method for brewery use.

Quantitative PCR (qPCR) usage has increased in brewery application because it allows the rapid quantitation of target DNA at an extremely sensitive level (such as from a single cell) (Bokulich et al., 2012a). The notable drawbacks include the high cost of the initial instrument and software, increased expertise required over conventional PCR, as well as concerns for quantitation accuracy given that the signal output does not discriminate living and non-living cells, and that results are influenced by the target gene copy number. Thus, an appropriately controlled and validated system is critical for drawing accurate conclusions. Since qPCR is not a community-profiling technique, it has limited use in interrogating mixed-culture fermentations or unknown isolates. Nonetheless, qPCR allows for the accurate monitoring of both the presence and quantity of specific populations in the brewing environment, and has notable advantages over other methods in terms of analysis speed and achievable sensitivity. Reverse transcription qPCR (RT-qPCR) assays have also been developed which analyse actively transcribed mRNA content, and thus viable cells, such as for the detection of the BSR LAB hop tolerance genes, though these assays must still be stringently and appropriately controlled (Bergsveinson et al., 2012; Sami et al., 1997b; Haakensen et al., 2007).

PCR assays continue to be optimized as the methodology itself evolves. A recent application is droplet digital PCR (ddPCR), which operates on the principle of absolute target quantification without need for internal control genes or excessive reaction replicates (Hindson et al., 2011; Pinheiro et al., 2012). ddPCR was recently used to investigate the copy number of hop tolerance genes within a brewery setting (Bokulich et al., 2015). Though ddPCR is limited in the number of targets it can interrogate, and by its cost, it most certainly can be further developed and applied to investigate gene target distribution and abundance within a brewery or contaminated sample (Hindson et al., 2011).

 
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