A DNA array based assay for the characterization of microbial community in raw milk

• Published in: Journal of microbiological methods Vol. 78; no. 2; pp. 181 - 188
• Main Authors: Giannino, Maria Laura, Aliprandi, Michele, Feligini, Maria, Vanoni, Laura, Brasca, Milena, Fracchetti, Fabio
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.08.2009; Elsevier
• Subjects: Animals; Bacteria; Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Bacterial Proteins - genetics; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biodiversity; Biological and medical sciences; Campylobacter jejuni; DNA, Bacterial - genetics; DNA, Ribosomal - genetics; Enterococcus faecalis; Escherichia coli; Fundamental and applied biological sciences. Psychology; Lactococcus lactis; Listeria monocytogenes; Microbiology; Milk - microbiology; Multiplex PCR; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide microarray; Polymerase Chain Reaction - methods; Raw milk; RNA, Ribosomal, 16S - genetics; RNA, Ribosomal, 23S - genetics; Salmonella; Streptococcus thermophilus; Virulence Factors - genetics; Yersinia enterocolitica; Raw milk; Multiplex PCR; Bacteria; Oligonucleotide microarray; Polymerase chain reaction; Multiplex polymerase chain reaction; DNA chip; Method; Detection; Microbial community
• ISSN: 0167-7012; 1872-8359; 1872-8359
• DOI: 10.1016/j.mimet.2009.05.015

An oligonucleotide array based on PCR method containing a combination of probes for taxonomic markers and species-specific virulence genes was developed for the simultaneous identification of 14 Lactic Acid Bacteria (LAB) and food-borne pathogenic bacteria in raw milk. The hypervariable regions V3 and V6 of 16S, together with a variable region of 23S rRNA genes and several genes specific for virulence factors were selected. Universal primers and multiplex PCR were used for the rapid differentiation of bacterial species and low concentration of specific pathogenic and spoilage bacteria were detected in milk. The dominant species such as Streptococcus thermophilus, Enterococcus faecalis, Lactococcus lactis, and Leuconostoc lactis were identified by indirect-labelling reactions based upon incorporation of amino-allyl dUTPs. The results regarding food-borne pathogens detection showed highly specific hybridisation patterns with the genomic DNA from Campylobacter jejuni, Escherichia coli, Salmonella thyphimurium, Listeria monocytogenes and Yersinia enterocolitica. A clear differentiation of dominant species present in a complex microbial community such as raw milk was achieved by the application of short oligonucleotide probes which discriminate sequences differing by few nucleotides.