Wu F M, Muriana P M
Department of Food Science, Purdue University, West Lafayette, IN 47907-1160, USA.
Int J Food Microbiol. 1995 Oct;27(2-3):161-74. doi: 10.1016/0168-1605(94)00162-y.
Genomic DNA from Listeria innocua and Listeria ivanovii was used in subtractive hybridization with DNA from Listeria monocytogenes involving two amplification strategies. Subtraction was accomplished by labelling the subtracting DNA with biotin and removal after liquid hybridization with tester DNA (L. monocytogenes) by reaction with streptavidin and phenol extraction. In one strategy, L. monocytogenes DNA was poly(A)-tailed with terminal transferase and amplified asymmetrically after subtraction. In another procedure, adapters ligated to the target DNA allowed symmetrical amplification after subtraction using an adapter-specific primer; in both amplifications, the amplified products were labelled with biotin-modified dUTP. Southern hybridization of the amplified/subtracted probes with tester- and subtractor-related strains demonstrated numerous L. monocytogenes-specific sequences. The genome-subtracted mixed probe identified 7 RFLP patterns among 13 strains of L. monocytogenes representing 11 L. monocytogenes serovars. Southern blot analysis demonstrated that the subtracted probe cross-hybridized to two bands among L. welshimeri strains but had little or no hybridization with five other species of Listeria including L. innocua, L. ivanovii, L. seeligeri, L. grayi, and L. murrayi. These data demonstrate that genomic subtraction via subtractive hybridization is a powerful method to enrich for specie-specific sequences in L. monocytogenes; the enriched sequences in the subtracted probe may be useful for typing L. monocytogenes strains by specific RFLP patterns or for cloning L. monocytogenes-specific sequences.
无害李斯特菌和伊氏李斯特菌的基因组DNA用于与单核细胞增生李斯特菌的DNA进行消减杂交，涉及两种扩增策略。通过用生物素标记消减DNA并在与检测DNA（单核细胞增生李斯特菌）进行液相杂交后，通过与抗生物素蛋白反应和苯酚抽提去除来完成消减。在一种策略中，单核细胞增生李斯特菌DNA用末端转移酶进行聚（A）加尾，并在消减后不对称扩增。在另一种方法中，连接到靶DNA的衔接子允许在消减后使用衔接子特异性引物进行对称扩增；在两种扩增中，扩增产物都用生物素修饰的dUTP进行标记。扩增/消减探针与检测相关菌株和消减相关菌株的Southern杂交显示出许多单核细胞增生李斯特菌特异性序列。基因组消减混合探针在代表11种单核细胞增生李斯特菌血清型的13株单核细胞增生李斯特菌中鉴定出7种RFLP模式。Southern印迹分析表明，消减探针与韦氏李斯特菌菌株中的两条带交叉杂交，但与其他五种李斯特菌，包括无害李斯特菌、伊氏李斯特菌、西尔李斯特菌、格氏李斯特菌和默氏李斯特菌几乎没有杂交或没有杂交。这些数据表明，通过消减杂交进行基因组消减是富集单核细胞增生李斯特菌物种特异性序列的有力方法；消减探针中富集的序列可能有助于通过特定的RFLP模式对单核细胞增生李斯特菌菌株进行分型或克隆单核细胞增生李斯特菌特异性序列。
