Detection of Gram-negative Bacteria in Broad-range PCR Amplifying 16S rRNA Gene with Semi-nested Primers and Its Application in Market Milk

16S rRNA 유전자의 Semi-nested Primer를 이용한 Broad-range PCR에 의한 그람음성세균의 검출과 시유에서의 응용

  • Choi, Suk-Ho (Dept of Biotechnology, Sangji University) ;
  • Choi, J.J. (Dept of Biotechnology, Sangji University) ;
  • Lee, S.B. (Dept of Biotechnology, Sangji University)
  • 최석호 (상지대학교 생명공학과) ;
  • 최정준 (상지대학교 생명공학과) ;
  • 이승배 (상지대학교 생명공학과)
  • Published : 2005.06.30


A two-step broad-range PCR method detecting gram-negative bacteria at the level as low as 2 CFU was developed by using primers of GNFI and GNRI and then semi-nested primer of GNF2 and GNRI. The nucleotide sequences of the primers were determined based on l6S rRNA gene. The DNA fragments of 1173 bp and 169 bp were amplified in one-step PCRs with primer sets of GNFI-GNRI and GNF2-GNRl, respectively, using template DNA from seven strains of gram-negative bacteria including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas spp., and Acinetobacter baumaii but not from Achromobacter lyticus, Alca/igens faecalis, and five strains of gram-positive bacteria. DNA fragments of 180 bp were amplified from LTLT-pasteurized milk and UHf-pasteurized milk in the two-step PCR. The DNA fragments were amplified from LTLT-pasteurized milk which was added with Pseudomonas j/uorescens and subsequently heated at 65 $^{\circ}C$, 80 $^{\circ}C$, and 100 $^{\circ}C$ for 30 min but they were not amplified from the milk autoclaved at 121$^{\circ}C$ for 15 min. It was suggested in PCR that Pseudomonas fluorescens heated at 65 $^{\circ}C$ for 30 min in milk was more sensitive to DNase treatment than viable bacteria.


PCR;16S rRNA;Gram-negative bacteria;Market milk


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