DIFFERENTIATION OF THE SPOILAGE YEAST ZYGOSACCHAROMYCES-BAILII FROM OTHER ZYGOSACCHAROMYCES SPECIES USING 18S RDNA AS TARGET FOR A NONRADIOACTIVE LIGASE DETECTION REACTION

A non-radioactive PCR coupled ligase detection reaction was developed to discriminate the food spoilage yeasts Zygosaccharomyces bailii and Z. bisporus from each other and from other members of the genus. A sho rt region of the 18S rRNA gene was amplified from boiled cell lysates and polymerase chain reaction (PCR) products used as target in the tem plate directed ligation of two adjacent oligonucleotides. Ligated prod ucts were captured using biotin-streptavidin chemistry and detected us ing digoxigenin immuno-chemiluminescence. The ligase detection reactio n was able to discriminate to the species level, targeting a single ba se deletion. The specificity of the reaction was assessed using seven species of the genus Zygosaccharomyces. Only strains of Z. bailii and Z. bisporus gave positive results with their respective primer sets. T he lower detection limit of the strategy was 10pg (3 x 10(7) targets) of amplified product.