title: Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.
abstract: DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.
authors.affiliation: International Centre for Brewing and Distilling, School of Life Sciences, Heriot- Watt University, Edinburgh EH14 4AS, UK.
authors.forename: A D
authors.initials: AD
authors.lastname: Paradh
authors.affiliation: International Centre for Brewing and Distilling, School of Life Sciences, Heriot- Watt University, Edinburgh EH14 4AS, UK.
authors.forename: A E
authors.initials: AE
authors.lastname: Hill
authors.affiliation: International Centre for Brewing and Distilling, School of Life Sciences, Heriot- Watt University, Edinburgh EH14 4AS, UK. Electronic address: W.J.Mitchell@hw.ac.uk.
authors.forename: W J
authors.initials: WJ
authors.lastname: Mitchell
mesh_terms: D001515:Beer / Q000382:microbiology*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005516:Food Microbiology / Q000379:methods*; D045851:Megasphaera / Q000235:genetics / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D045852:Pectinatus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D004275:DNA, Ribosomal
keywords: Acridinium ester; Hybridisation protection assay; Megasphaera; Pectinatus