Alanine racemase gene fragments as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods

Yoko Okubo, Kumio Yokoigawa, Hiroyasu Kawai
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Abstract

Alanine racemase gene fragments containing non-conserved regions of the gene were evaluated as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods. The gene fragments were amplified from genomic DNA of each bacterium by polymerase chain reaction with degenerate oligonucleotide primers, and labeled with digoxigenin as probes for detecting each bacterium. Foods and bacteria were treated at 25°C for 10 min in 0.1 N NaOH containing 0.5% SDS before being directly spotted onto nylon membranes for DNA hybridization. When the specificities of the probes were analyzed using a total of 86 strains (23 genera and 65 species) of bacteria including 29 Bacillus strains (20 species), each probe was specific for the respective target bacteria. A variety of foods inoculated with B. stearothermophilus or B. psychrosaccharolyticus were positive as determined by hybridization with the respective probe, whereas uninoculated foods were negative. The alanine racemase gene fragments could be used as specific probes for detecting B. stearothermophilus and B. psychrosaccharolyticus in foods.

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丙氨酸消旋酶基因片段在食品中嗜脂嗜热芽孢杆菌和嗜冷糖芽孢杆菌检测中的应用
研究了含有非保守区域的丙氨酸消旋酶基因片段作为检测食品中嗜热脂肪芽孢杆菌和嗜冷糖芽孢杆菌的探针。用聚合酶链反应从每个细菌的基因组DNA中扩增出基因片段,并用地高辛标记作为检测每个细菌的探针。食品和细菌在含0.5% SDS的0.1 N NaOH中25°C处理10分钟,然后直接定位到尼龙膜上进行DNA杂交。对包括芽孢杆菌29株(20种)在内的86株(23属65种)细菌进行特异性分析时,每种探针对各自的目标细菌都具有特异性。通过与探针杂交确定,接种嗜脂嗜热B.菌或嗜糖嗜冷B.菌的各种食物呈阳性,而未接种的食物呈阴性。丙氨酸消旋酶基因片段可作为食品中嗜脂嗜热双歧杆菌和嗜冷解糖双歧杆菌的特异性探针。
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