• Journal of Applied Biological Chemistry
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• 제44권4호
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• pp.163-166
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• 2001

Piezoelectric (PZ) crystal biosensor system was used to detect the DNA of food pathogenic Listeria monocytogenes. L. monocytogenes-specific DNA was multiplied via the polymerase chain reaction using LM1 oligonucleotide (5'-TTACGAATTAAAAAGGAGCG-3') and LM2 oligonucleotide (5'-TTAAATCAGCAGGGGTCTTT-3') as primers. DNA fragment of 161 bp, which was specific only for L. monocytogenes, was observed. To obtain a large amount of single-stranded DNA containing an SH group used for coupling to the gold electrode chemisorptively, LM1 oligonucleotide containing a mercaptohexyl group was utilized as a single strand PCR primer. The PCR product was immobilized onto the gold electrode of PZ crystal, and hybridization was monitored in quartz crystal microbalance (QCM) system by injecting the antisense single-stranded DNA of 161 nucleotides obtained via the single strand PCR using the unmodified LM2 primer. Approximately 70 Hz of frequency drop was observed in the QCM system in the case of two consecutive injections of $5{\mu}g$ of the antisense single-stranded DNA.

• 한국식품과학회지
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• 제31권6호
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• pp.1628-1634
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• 1999

Listeria monocytogenes의 식품 속에서 신속하고 특이적인 검출을 위하여, listeriolysin O gene에 의한 primer, LM 1과 LM 2를 선택하여 PCR을 수행하였다. L. monocytogenes의 DNA 추출이나 cell lysis 없이 intact whole cell을 직접 이용하여 PCR을 하였으며 $10^{2-6}$ CFU 수준의 균체 배양액으로부터 L. monocytogenes에 특이적인 702 bp의 PCR 증폭 산물을 확인하였다. 우유, 닭고기, 김치 등의 식품에 L. monocytogenes를 접종하여 증균배양 전후 균의 PCR에 대한 감도와 생균수를 비교한 결과, 실험된 식품 속에서의 L. monocytogenes의 검출 감도는 순수 배양액에서의 경우에 비해 약 1/10로 둔화되었으나 역시 특이적인 검출이 가능하였다. Primer LM 1과 2를 이용한 본 실험조건에서의 L. monocytogenes의 PCR에 의한 검출은 약 4시간으로 확인이 가능하였으며, 기존의 배양 방법에 비해, 특이성이나 검출 속도 면에서 식품위생 실무에 적용하기 위한 높은 잠재력을 보여 주었다.

• 한국미생물·생명공학회지
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• 제34권1호
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• pp.23-27
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• 2006

본 연구에서는 국내시장에서 유통되는 육류, 냉동식품, 생우유, 조개류 등과 같은 식품으로부터 L. monocytogenes들을 분리하고 혈청형을 결정하였다. RAPD 결과를 통해서 L. monocytogenes는 Listeria 속의 다른 균주들과 전체 밴드의 수와 크기에서 구별이 되었다. 또한, L. monocytogenes는 2개 그룹으로 구별되었으며, 그룹 I은 L. monocytogenes 1/2b, 4e, 4b, 그룹 II는 L. monocytogenes 1/2a, 1/2c, 3 혈청형 그룹별로 분류되었다. 결론적으로 RAPD 방법과 serotyping은 L. monocytogenes를 분류하는 데에 있어서 기존의 방법의 단점을 보완한 새로운 가능성을 제시하였다.

• 대한수의학회지
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• 제43권2호
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• pp.231-237
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• 2003

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

• 대한수의학회지
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• 제49권4호
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• pp.319-328
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• 2009

This study investigated the epidemiology of Listeria (L.) monocytogenes, a food-borne pathogen. The epidemiology of food-borne pathogens is of great importance for clarifying bacterial origin and preventing bacterial contamination and infection. This work examined 68 L. monocytogenes strains, including 11 reference strains and 57 isolates from imported US beef, domestic meats (beef, pork, chicken meat), raw milk, and milk plants. The random amplified polymorphic DNA (RAPD) techniques were optimized to develop a standard molecular epidemiological analysis of L. monocytogenes. There was great genetic variability among the isolates, which produced 24 and 34 RAPD patterns with primer HLWL85 and HLWL74, respectively. The discriminatory power of the RAPD methods with HLWL85 and HLWL74 primer were very high (DI = 0.957; S ${\geq}$ 80%, S ${\geq}$ 95%). Some RAPD types were specific to origin. A few RAPD types were specific for L. monocytogenes strains belonging to a particular serotype. Using the HLWL85 primer, the strains isolated from milk plants could be distinguished from the other strains. And using the HLWL74 primer, the strains isolated from imported beef (US) could be distinguished completely from the other strains.

• Journal of Microbiology
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• 제44권1호
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• pp.92-97
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• 2006

We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to $0.05\;pM/{\mu}l$. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1276-1284
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• 2006

Listeria monocytogenes is a well-known high-risk foodborne pathogen that grows at refrigeration temperature and is responsible for outbreaks of listeriosis. We report here the incidence of L. monocytogenes in fresh chicken carcasses and present genetic diversity of L. monocytogenes isolates. In this study, 25 g of chicken carcasses from markets in Korea were examined according to the FDA method, and presumptive isolates were confirmed by multiplex PCR assay. L. monocytogenes isolates were analyzed by Pulsed-Field Gel Electrophoresis using restriction enzymes, ApaI and AscI, to obtain strain-specific DNA fragments profiles. Antimicrobial resistance of L. monocytogenes strains against generally used antibiotics (Penicillin G, Kanamycin, Tetracycline, Vancomycin, Cephalothin, Rifampicin, Erythromycin, Ampicillin, Gentamicin, Streptomycin, and Chloramphenicol) were analyzed by NCCLS protocols to examine the presence of antimicrobial resistance in natural L. monocytogenes. Of a total 274 chicken samples, 81 samples (29.6%) were positive for L. monocytogenes. Listeria innocua (50.1%), Listeria welshimeri (6.9%), and Listeria grayi (11.3%) were also detected. PFGE analysis, using restriction enzymes ApaI and AscI, showed 27 pulsotypes of L. monocytogenes. Antimicrobial resistance analysis confirmed the existence of antimicrobial resistance for penicillin G and tetracycline in isolated L. monocytogenes strains.

• 한국식품위생안전성학회지
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• 제18권1호
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• pp.25-29
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• 2003

리스테리아의 p60단백질은 Listeria속 고유의 단백질로써, 주요 세포외 단백질이기에, 식품에서 이 세균의 존재를 밝혀주는 중요한 지시단백질로 사용된다. 본 연구는 재조합 DNA 방법으로 재조합-p60 단백질을 대장균에서 생산하였으며, 아밀로오스 컬럼 크로마토그라피의 방법으로 정제하여, Listeria spp.의 p60에 특이적 항체를 생산하는 단일클론을 선별하였다. 생산된 항p60 항체 1H4는 병원성 Listeria 균주인 L. monocytogenes, L. ivanovii, L. weishi meri II 특이적으로 강한 항체반응을 보였으며, Listeria속의 비병원성균인 L. innocua등과는 상대적으로 매우 약한 항체반응을 보였으며, 타 세균의 단백질과는 거의 반응하지 않았다. 1H4항체는 복수생산법에 의해 대량생산되었으며, 이 항체의 특이성은 면역학적 방법에 의한 리스테리아 검출키트의 개발에 유용하게 사용될 수 있을 것이다.

• 한국축산식품학회지
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• 제34권5호
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• pp.717-723
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• 2014

A rapid and specific PCR assay for the simultaneous detection of Listeria monocytogenes, Escherichia coli O157:H7, Bacillus cereus, Salmonella spp., and Staphylococcus aureus in foods was developed to reduce the detection time and to increase sensitivity. Multiplex PCR developed in this study produced only actA, fliC, hbl, invA, ileS amplicons, but did not produce any non-specific amplicon. The primer sets successfully amplified the target genes in the multiplex PCR without any non-specific or additional bands on the other strains. The multiplex PCR assays also amplified some target genes from five pathogens, and multiplex amplification was obtained from as little as 1 pg of DNA. According to the results from the sensitivity evaluation, the multiplex PCR developed in this study detected 10 cells/mL of the pathogens inoculated in milk samples, respectively. The results suggested that multiplex PCR was an effective assay demonstrating high specificity for the simultaneous detection of five target pathogens in food system.

• Journal of Microbiology and Biotechnology
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• 제30권2호
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• pp.259-270
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• 2020

Listeria monocytogenes is a gram-positive, facultative anaerobe food pathogen responsible for the listeriosis that mostly occurs during the low-temperature storage of a cold cut or dairy products. To understand the systemic response to a wide range of growth temperatures, L. monocytogenes were cultivated at a different temperature from 10℃ to 42℃, then whole cell proteomic analysis has been performed both exponential and stationary cells. The specific growth rate increased proportionally with the increase in growth temperature. The maximum growth rate was observed at 37℃ and was maintained at 42℃. Global protein expression profiles mainly depended on the growth temperatures showing similar clusters between exponential and stationary phases. Expressed proteins were categorized by their belonging metabolic systems and then, evaluated the change of expression level in regard to the growth temperature and stages. DnaK, GroEL, GroES, GrpE, and CspB, which were the heat&cold shock response proteins, increased their expression with increasing the growth temperatures. In particular, GroES and CspB were expressed more than 100-fold than at low temperatures during the exponential phase. Meanwhile, CspL, another cold shock protein, overexpressed at a low temperature then exponentially decreased its expression to 65-folds. Chemotaxis protein CheV and flagella proteins were highly expressed at low temperatures and stationary phases. Housekeeping proteins maintained their expression levels constant regardless of growth temperature or growth phases. Most of the growth related proteins, which include central carbon catabolic enzymes, were highly expressed at 30℃ then decreased sharply at high growth temperatures.