Oligonucleotide-direct epifluorescent filter technique

PCR the in situ system has no problem with inhibitory effects from the beer matrix as no polymerase enzyme is needed. 

There are a number of different probes available, including detection probes, which are labelled with a fluorescence marker or, for example, digoxigenin for a further linkage with an antibody-enzyme complex and then a later colorimetric reaction with a chromogenic substrate (such as nitro blue tetrazolium for alkaline phophatase) (Helentjaris McCreery, 1996 Kempf et al., 2000), Capture probes are used to bind the target sequence (RNA or DNA) to a plate or another surface. In most cases the probes are labelled with biotin to react with avidin, which is coated on a plate (Riley, Marshall, Coleman, 1986). 

2 rRNA as detection target (application, theory to RNA, RNase, live and dead cells, probes, sample preparation, comparison to PCR) 

For the detection of bacteria and other organisms it is interesting using rRNA as a detection target as RNA in normal cases only exists in living cells and also in numerous copies (up to several thousands of ribosomes per bacteria (Kaczanowska Ryd6n-Aulin, 2007) and for yeast even more, close to 200,000 (Warner, 1999)). PCR methods, for example, also detect DNA of dead cells as double-stranded DNA is quite stable while the single-stranded RNA is decomposed within a few hours from nucleases. Because of the numerous ribosomes in a bacteria or yeast cell there is no need to do a PCR and a direct detection of bacteria or yeasts is possible. 

There are three classical FISH detection kits available on the market called VIT -beer (Vermicon), which detects the beer spoiling organisms. The protocol of the method takes no more than 3h and can be directly used for isolates or for beer samples after an enrichment step. One kit detects all members of beer-spoilage lactic acid bacteria (red fluorescence) and additional specific Lactobacillus brevis (red and green fluorescence), the most prominent beer spoiling organism. With another kit it is possible to 

---