Application of subtractive hybridization towards the isolation of species-specific DNA probes for Listeria monocytogenes
Abstract
Genomic DNA from Listeria innocua and Listeria ivanovii was used in subtractive hybridization with DNA from Listeria monocytogenes involving two amplification strategies: asymmetrical and symmetrical amplifications. Subtraction was accomplished by labeling the subtracting DNA with biotin and removal after liquid hybridization with tester DNA (L. monocytogenes) by reaction with streptavidin and phenol extraction. The resulting subtracted probe was labeled with biotin-modified dUTP in a PCR amplification. Southern hybridization of the amplified/subtracted probes with tester- and subtracter-related strains demonstrated numerous L. monocytogenes-specific sequences. When tested on 24 Listeria strains (7 species) and 13 strains of other bacterial genera, this probe was specific to L. monocytogenes except two cross-hybridization bands with L. welshimeri. The mixed probe was tested on 16 strains of L. monocytogenes isolated from 6 brands of hot dogs. Analysis of restriction fragment length polymorphisms (RFLP) revealed 11 patterns among 16 strains of L. monocytogenes tested, indicating a high degree of sequence heterogeneity in L. monocytogenes and that this mixed probe is potentially useful for the differentiation of L. monocytogenes strains. Individual probe DNA fragments were cloned by ligation of gel-eluted EcoRI-digested L. monocytogenes genomic DNA or by SalI-digested PCR products onto a plasmid vector. Five large genomic DNA fragments and 90 PCR clones were obtained. Sequence analysis of the PCR clones identified 21 unique DNA fragments. Two hly A, two ATPase, three fru II enzyme and one lex A gene fragments were identified. These individual DNA fragments were, again, tested on 13 L. monocytogenes strains from various sources for their specificity. Two clones, representing partial hly A gene fragments, were specific to all the L. monocytogenes strains tested. Several other clones with either putative or unknown functions also showed specificity to as many as 11 out of 13 L. monocytogenes strains. A combination of DNA fragments other than the hly A gene could potentially detect all of the L. monocytogenes strains tested. The isolated L. monocytogenes-specific DNA fragments are potentially useful in designing PCR primers for specific PCR detection of L. monocytogenes in food.
Degree
Ph.D.
Advisors
Muriana, Purdue University.
Subject Area
Food science|Microbiology|Molecular biology
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