16S rRNA 유전자의 Semi-nested Primer를 이용한 Broad-range PCR에 의한 그람음성세균의 검출과 시유에서의 응용
Detection of Gram-negative Bacteria in Broad-range PCR Amplifying 16S rRNA Gene with Semi-nested Primers and Its Application in Market Milk
0
조회
0
다운로드
0
인용
0
참고문헌
초록
Semi-nested primer들을 사용하여 그람음성세균을 2 CFU까지 검출할 수 있는 2단계 broad- range PCR을 개발하였다. 첫 번째 PCR에서는 GNF1과 GNR1를 primer로서 사용하였으며 두 번째 PCR에서 GNF2와 GNR1를 semi-nested primer로서 사용하였다. GNF1-GNR1 및 GNF2- GNR1 primer 쌍을 사용한 두개의 1단계 PCR 모두에서 Escherichia coli, Enterobacter aero- genes, Klebsiella pneumoniae, Pseudomonas spp. 및 Acinetobacter baumaii를 포함한 그람음성세균7균주에서 16S rRNA 유전자 절편이 검출되었고 Achromobacter lyticus와 Alcaligens faecalis, 및 그람양성세균 5균주에서는 검출되지 않았다. DNA를 신속하게 추출하는 방법 중에서 가열법으로 Pseudomonas fluorescens의 DNA를 추출한 경우에서 semi-nested primer를 사용한 2단계 PCR에 의해 2CFU까지 검출할 수 있었다. 초고온 순간살균유와 저온장시간 살균유로부터 two-step PCR에 의해 16S rRNA 유전자의 DNA가 검출되었다. 저온장시간 살균유에 P. fluorescens로 첨가한 후에 65℃, 80℃ 100℃에서 30분간 가열한 시료에서 16S rRNA가 검출되었다. 그러나 121℃에서 15분간 autoclave한 시료에는 검출되지 않았다. P. fluorescens를 첨가한 시유를 65℃에서 30분간 가열처리한 시료가 가열처리하지 않은 시료에 비해 DNase 처리에 대해 더 민감하게 DNA가 파괴되어 2단계 PCR에 의해 16S rDNA가 검출되지 않았다.
Abstract
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 GNF1 and GNR1 and then semi-nested primer of GNF2 and GNR1. The nucleotide sequences of the primers were determined based on 16S rRNA gene. The DNA fragments of 1173 bp and 169 bp were amplified in one-step PCRs with primer sets of GNF1-GNR1 and GNF2-GNR1, 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, Alcaligens faecalis, and five strains of gram-positive bacteria. DNA fragments of 180 bp were amplified from LTLT-pasteurized milk and UHT-pasteurized milk in the two-step PCR. The DNA fragments were amplified from LTLT-pasteurized milk which was added with Pseudomonas fluorescens and subsequently heated at 65℃, 80℃, and 100℃ for 30 min but they were not amplified from the milk autoclaved at 121℃ for 15min. It was suggested in PCR that Pseudomonas fluorescens heated at 65℃ for 30 min in milk was more sensitive to DNase treatment than viable bacteria.
참고문헌 (22)
- "Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods", pp.597~600 (1991)
- "Detection of multiple virulence-associated genes of Listeria monocytogens by PCR in artificially contaminated milk samples", pp.3023~3026 (1994)
- "Salmonella DNA persistence in natural eawaters using PCR analysis", 82, pp.507~510 (1997)
- "Phylogenetic group-specific oligonucleotide probe for identification of single microbial cells", giovan(i), pp.720~726 (1988)
- "PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria including bacteria found in cerebrospinal fluid", pp.335~351 (1994)
- "Development of nested PCR based on the ViaB sequence to detect Salmonella typhi", 33, pp.775~777 (1995)
- "Detection of viable and dead Listeria monocytogenes by PCR", 14, pp.103~110 (1997)
- "Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers", 61, pp.817~819 (1995)
- "Bergey’s manual of systematic bacteriology" (1984), Williams and Wilkins
- "Catalogue of tests for the detection of post-pasteurization contamination of milk Bulletin of the IDF 281", pp.13~34 (1993)
- "Analysis of DNA extract from formalin-fixed paraffin-embedded tissues by enzy- matice amplification and hybridization with sequence- specific oligonucleotides", pp.710~6 (1987)
- "Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxi- genic Escherichia coli by the polymerase chain reaction J", 77, pp.73~9 (1994)
- "rRNA stability in heat-killed and UV- irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157", 64, pp.4264~4268 (1998)
- "Mitochondrial DNA sequences from a 7000-year brain", pp.0775~87 (1988)
- "Detection of Yersinia enterocolitica 0 3 in faecal samples and tonsil swabs from pigs using IMS and PCR", 78, pp.563~568 (1995)
- "Direct detection of viable bacteria and yeasts by reverse transcriptase PCR in contaminated milk samples after heat treatment", 64, pp.1157~1160 (1998)
- "Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes", pp.6823~31 (1991)
- "Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction", pp.3443~3447 (1992)
- "Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay", pp.2743~2745 (1993)
- "Bacterial evolution", 51, pp.221~271 (1987)
- "Methods for the detection quantification and and differentiation of infectious versus non-infectious pathogens in a sample" (2001)
- "시유의 2차오염과 저장가능기간을 결정하기 위한 resazurin 환원시간검사", 46, pp.999~1006 (2004)