본 연구는 S. aureus을 신속하게 검출할 수 있는 PCR primer를 개발하고자 실시되었다. S. aureus를 포함한 Clostridium, Escherichia, Salmonella, Shigella, Vibrio, Bifidobacteria, Lactobacillus, Streptococcus 등 그람 양성 및 그람 음성 균주 17 종의 genomic DNA를 대상으로 20종의 랜덤 primer들을 사용하여 PCR을 실시하였다. S. aureus의 특이적 밴드를 선발하여 DNA 염기 서열을 분석하였고 이를 바탕으로 5쌍의 primer를 제작하였으며 그 중 primer SYU-5를 사용하여 PCR을 실시할 경우, 단일 밴드로 나타나는 다른 균주들에 비해 S. aureus 균주에서 약 250, 550 및 850 bp 크기의 세 개 밴드들이 검출되었다. 또한, S. aureus가 첨가된 8종 균주들의 genomic DNA 혼합 용액과 각종 유제품 및 분쇄육을 대상으로 primer SYU-5의 특이적 검출능을 확인하였다. 본 연구에서 사용한 PCR 기법은 기존의 선택배지 배양법과 비교하여 시간 및 노동력 측면에서 식중독 유발 S. aureus의 검출에 보다 효과적인 방법으로 사용 가능하였다. 또한, 본 연구의 결과는 다양한 병원균들의 특이적 검출을 위한 PCR 기법 개발에 기초 자료를 제공할 수 있다.

본 연구는 들깻잎과 들깻잎 생산 환경을 대상으로 S. aureus를 분리하고 MLVA를 이용해 분리된 S. aureus의 오 염경로를 분석하였다. 그 결과, 들깻잎에서 S. aureus의 검출빈도는 7.9% (9/114)였고 검출수준은 0~2.92 log10 CFU/ g였으며 2곳의 농가에서 S. aureus가 2.00 log10CFU/g 이상으로 검출되었다. S. aureus가 분리된 5농가를 대상으로 들깻잎과 직접 접촉하는 시료에서 S. aureus를 분리하였으며 5 농가 중 3농가의 작업환경 (농가의 포장대, 포장비 닐, 작업자 손, 복장 등)에서 S. aureus가 검출되었다. 들 깻잎과 생산 환경에서 분리된 S. aureus를 대상으로 MLVA 를 수행했을 때 포장대, 작업자의 손, 포장비닐 및 관개용 수에서 분리된 균주와 들깻잎에서 분리된 S. aureus가 동일한 패턴을 보여 들깻잎의 오염은 이들 시료와 연관되어 있을 것으로 추정된다. 따라서 MLVA를 이용한 유전자 typing은 농산물이나 식품 중 S. aureus의 오염원을 신속 하고 경제적으로 구명하는데 활용할 수 있을 것으로 판단 된다.

The objective of this study was to investigate the antimicrobial effects of carvacrol (CV) against Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) strains in milk. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of CV against S. aureus and E. coli O157:H7 were determined. In addition, bactericidal kinetics and antimicrobial activity of CV against the aforementioned pathogens in milk over a period of 2 weeks were investigated. CV exhibited antibacterial activity against both foodborne pathogens tested. The MIC and MBC of CV against S. aureus were 15.0 and 20 mg/mL, respectively, whereas those against E. coli O157:H7 were 16.0 and 32 mg/mL, respectively. In time-kill assays, CV at MBC reduced the number of S. aureus and E. coli O157:H7 in milk to undetectable levels within 24 hr. The antibacterial effects of CV persisted for 14 days without any loss of activity. Results of this study suggest that CV has a potential antibacterial activity against foodborne pathogens such as S. aureus and E. coli O157:H7 in milk.

Lacticin NK34 is a small nisin-like bacteriocin present in the supernatants of an isolate of Lactococcus lactis from jeotgal, a salted and fermented Korean food made with seafood such as shrimp, oysters, and fish. Recently, we demonstrated that a partially purified NK34 is highly effective against various Staphylococcus species in a murine infection model. In this study, the two major bacterial pathogens associated with bovine mastitis, Streptococcus aureus and S. agalactiae, were evaluated for their susceptibility to NK34 in vitro using a standard teat-dip assay as well as in vivo using mastitic cows infected with one of these pathogenic strains. The experimental analyses showed a significant decrease (up to 98 times) in the bacterial numbers between the NK34-treated and -untreated teats. Moreover, a dramatic reduction in somatic cell counts was observed at 3 days post-treatment with 10 ml of NK34 administered directly into the mastitic cows. Neither S. aureus nor S. agalactiae were recovered. Taken together, these results imply that lacticin NK34 is an alternative antimicrobial substitute for the treatment of bovine mastitis, caused especially by either S. aureus or S. agalactiae.

목적: 황색포도상구균(Staphylococcus aureus)과 녹농균(Pseudomonas aeruginosa)으로 오염시킨 RGP 렌즈를 3가지 종류의 다목적용액에 침윤시킨 후 각 시간대별 두 균주의 항균효과를 비교하여 렌즈 소독에 필요한 침윤시간을 제시하고자 한다. 대상과 방법: 황색포도상구균과 녹농균의 배양액(농도 109 Colony Forming Unit/ml) 0.1ml와 LB broth 0.9ml 혼합액에 RGP 렌즈를 넣은 후 24시간 배양 후 실험을 시행하였다. 3가지 종류의 다목적용액을 1시간, 2시간, 3시간, 4시간, 5시간, 6시간, 7시간 동안 침윤시켜 항균 효과를 비교하였다. 결과: 황색포도상구균과 녹농균에 대한 다목적용액 간에 항균효과 차이는, 시간대별로 1시간 침윤으로 전체 균의 cfu가 약 43~47%, 55~63% 감소하였고, 2시간 침윤으로는 약 77~80%, 77~86% 감소하였다. 3시간 침윤으로는 약 97~98%, 85~97% cfu가 감소하였고, 4시간 침윤으로는 100%, 95~100% cfu가 감소하였다. 그리고 5시간, 6시간, 7시간 침윤으로는 다목적용액 모두 황색포도상구균(Staphylococcus aureus)과 녹농균(Pseudomonas aeruginosa)이 검출되지 않았다. 결론: 황색포도상구균은 다목적용액 4시간 이상의 침윤시간에서 검출되지 않았고 녹농균은 다목적용액 5시간 이상의 침윤시간에서 세균이 검출되지 않았다. 따라서 본 연구에 사용된 다목적용액을 RGP 콘택트렌즈 소독에 사용하는 경우 5시간 이상 침윤시켜야 할 것으로 사료된다.

본 연구는 20% ortho-phenylphenol을 함유한 훈증소독제, Fumagari OPP®의 Staphylococcus aureus (S. aureus)에 대한 살균효과를 평가하기 위해, French standard NF T 72-281에 따라 수행하였다. 배양 현탁액 중 S. aureus의 균수 (N 값), 훈증소독제에 노출된 각 담체의 균수 (n1, n2, n3), 평판배지법에 의한 시험균주 현탁액 중 균수 (N1), 여과법에 의한 시험균주 현탁액 중 균수 (N2), 그리고 대조 담체의 회복 균수의 평균값 (T 값)을 예비실험을 통해 구하였다. 또한, 훈증소독제에 노출된 S. aureus의 감소 균수 (d 값)는, T 값, 회복액 중 균수의 평균값 (n'1) 그리고 배지의 담체에서 증식한 균수의 평균값 (n'2) 등을 이용하여 산출 하였다. N 값은 4.0 × 108 CFU/mL이었으며, n1, n2, n3은 각각 0.5N1, 0.5N2, 0.5N1 보다 높게 나타났다. 그리고 T 값은 3.4 × 106CFU/mL이었다. 훈증소독제의 살균효과에 있어서, d 값은 6.43 logCFU/mL이었다. 훈증소독제에 대한 프랑스 기준에 따르면, 효과적인 살균력을 갖는 훈증 소독제의 d 값이 5 logCFU/mL 이상이어야 하는 것으로 규정하고 있다. 본 연구의 결과로부터, Fumagari OPP®는 S. aureus에 대해 높은 살균효과를 갖는 것으로 나타나, 병원성 세균으로 오염된 식품재료와 주방기기의 소독에 적용할 수 있을 것으로 사료된다.

본 연구는 가공치즈에서 Staphylococcus aureus의 생장 을 예측하기 위한 수학적 모델을 개발하였다. 모짜렐라 슬라이스 치즈와 체다 슬라이스 치즈에 S. aureus 혼합균액 (ATCC13565, ATCC14458, ATCC23235, ATCC27664, NCCP10826) 0.1 ml (log CFU/g)을 접종한 후 4oC (1440 h), 15oC (288 h), 25oC (72 h), and 30oC (48 h)에 저장하면서 총 세균 수와 S. aureus 세균 수를 tryptic soy agar와 mannitol salt agar를 이용해 각각 확인하였다. S. aureus의 세균 수 를 Baranyi model로 분석하여 생장률(μmax; log CFU·g−1·h−1), 유도기(LPD; h), 초기 세균수(log CFU/g), 최대 생장 세균 수(log CFU/g)를 계산함으로써 1차 모델을 개발하였다. 또 한 저장온도와 S. aureus의 μmax, LPD의 관계를 분석하기 위해 square root model과 exponential decay model을 이용 하였고 이를 통해 2차모델을 개발하였으며 개발된 모델의 평균제곱근 편차(RMSE)를 계산하여 적합성을 검증하였다. 4oC에서는 모든 가공치즈에서 황색포도상구균의 생장이 관찰되지 않았으나 15oC, 25oC, 30oC에서는 모짜렐라 슬라이스와 체다 슬라이스 치즈에서 황색포도상구균이 생장 하였으며(R² = 0.785-0.996) 저장온도가 높아짐에 따라 생장률은 증가한 반면 유도기는 감소하였다(R² = 0.879-0.999). 또한 개발된 모델의 RMSE 값은 0.3500-0.5344로 적합하였다. 따라서 본 연구결과는 가공치즈에서 황색포도상구균의 생장 예측에 유용하게 사용될 것이다.

표준 평판배지 정량검출법과 TEMPO STA를 이용한 황색포도상구균 정량 분석에 대한 상관성과 유의성을 평가 하기 위해 복합조리식품에 황색포도상구균을 인위적으로 접종하여 결과값을 통계 처리하여 유의성을 조사한 결과 두 실험방법간에는 유의적 차이가 없었다(p < 0.05). 상관 계수(r2)는 0.9672로 두 실험간에는 높은 연관성을 보였다. TEMPO STA를 466개의 시료에 적용한 결과 454개 (97.4%)의 시료는 문제가 없었으나 이중 12개의 시료에서는 error가 발생하였다. 그러나 TEMPO법은 실험 단계 간편하고 사용이 쉬우며 신속한 결과를 얻을 수 있으며 자동화된 장비로 실험 과정에 의한 오류를 최소화 할 수 있는 장점이 있으며 표준시험법인 표준 평판 배지법과도 유의적인 차이가 없어 정확한 정량검출이 가능할 것으로 생각된다. 최근 식품의 안전성확보를 위해 검사 품목이 다양해지며 수도 증가하는 추세이기 때문에 황색포도상구균의 정량 검사가 정확하고 신속해야 할 필요가 있으므로 TEMPO STA법의 필요성은 앞으로 크게 늘어날 것으로 보인다.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important causative microbes for nosocomial infe- ction and has been isolated from the dental environment. The purpose of this study was to investigate the antimi- crobial activity of linalool and α‐terpineol against MRSA isolates from a Korean population. In the experiments, we determined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these two compounds against 18 strains of MRSA. The data re- vealed that the MIC90/MBC90 values of linalool and α‐ terpineol against MRSA were >12.8 mg/ml and 6.4 mg/ml, respectively. These results indicate that α-terpineol has more potent antimicrobial activity against MRSA than lina- lool and may have utility as an anti‐MRSA cleansing agent for dental instruments and dental unit chairs.

The dual effectiveness of Opuntia ficus indica extracts for browning inhibition and microbial inactivation on fresh-cut apples was investigated. Prepared apple slices were treated with 25, 50, 100, 200 mg/mL Opuntia ficus indica extracts, packaged in polyethylene bags, and stored for 10 days at 4, 21oC. Results indicate that Opuntia ficus indica extracts significantly (P < 0.05) inhibited the browning reaction of fresh-cut apples. This treatment also reduced peroxidase activities. The populations of Staphylococcus aureus significantly decreased with increasing extract concentration (p < 0.05). In particular, S. aureus was reduced to non-detectable levels after 2 days in 100 mg/mL treatment at 4oC and 21oC. Opuntia ficus indica extracts therefore have antibacterial and antibrowning effects. The results suggest that Opuntia ficus indica extracts could be useful as a natural food preservative.

Saliva from the oral cavity was collected from 129 patients with periodontitis and 54 Staphylococcus aureus isolates were isolated from these samples. The S. aureus isolates were tested for their resistance patterns against 15 antibiotics using the disc diffusion method. The percentage of S. aureus isolates resistant to ampicillin was the highest (92.6%). In addition, the percentage of S. aureus isolates resistant to penicillin, oxacillin, cefotetan, cefepime and erythromycin was 90.7%, 11.1%, 11.1%, 9.3% and 5.6%, respectively. However, S. aureus isolates were susceptible to gentamycin, clindamycin, trimethoprim/sulfamethoxazole and vancomycin. 96.3% of S. aureus isolates were resistant to antibiotics. Among them, the percentage of two antibiotic-resistant S. aureus isolates was 74.1%, the percentage of one antibiotic-resistant S. aureus isolates was 5.6%, the percentage of three antibiotic-resistant S. aureus isolates was 3.7% and the percentage of S. aureus isolates resistant to more than 4 antibiotics was 13%. The most common multiple antimicrobial resistance pattern was AM-P. Furthermore, the percentage of methicillin-resistant S. aureus isolates was 11.1% and they were resistant to more than 3 antibiotics.

The purpose of this study is to evaluate antimicrobial effects of organic acid and some natural occurring antimicrobials against Staphylococcus aureus isolated from various origins (vegetables, peanut, pea leaf, kim- bab, person, perilla leaf, and animal) and to calculate their MIC and MBC values. Five organic acids (acetic, lactic, citric, malic, and propionic acid), three essential oils (carvacrol, thymol, and eugenol), and two other natural antimicrobials (nisin and cinnamic acid) were evaluated for their antimicrobial effects against 113 strains of S. aureus using combination treatments. Propionic acid (7%), nisin (1%), thymol (1%), carvacrol (1%) showed antimicrobial activities against S. aureus strains in agar disc diffusion test. And, carvacrol, thymol, and nisin were found to be the most effective with the lowest MIC values of 0.0313%, 0.0625%, and 0.0625% against S.aureus, respectively. Propionic acid (0.2313%) and citric acid (0.6000%) were the most effective among organic acids tested. Therefore, these five antimicrobials were selected for next combination treatments. Combination of propionic acid and citric acid were showed the strongest inhibitory effectiveness against S. aureus among combination treatments. These results suggest that organic acid such as propionic and citric acid, and natural occurring antimicrobial such as nisin, carvacrol, and thymol might be possibly used as preservatives for inhibiting S. aureus in foods.

The dental infections have been increased due to the widespread overuse and exposure to antibiotics. One of the well-known pathogens is S. aureus The pathogenic properties of S. aureus is associated with the virulence gene. The objective of this study is to investigate the distribution of virulence gene of S. aureus which may contribute to the prevention and treatment of bacterial infections. 54 strains of S. aureus were separated from saliva taken from 129 outpatients diagnosed with periodontitis from Jun. to Dec., 2010 in Seoul. 44 (46.6%) and 13 (31.7%) strains of them were obtained from 88 and 41 outpatients from the S and E dental clinics, respectively. Then, the distribution of virulence gene and genetic diversity were analyzed with the PCR technique. The result of the S. aureus isolates possessed coagulase gene and showed six polymorphism types 390~470 bp (1.8%), 550~633 bp (3.7%), 630~714 bp (9.3%), 715~795 bp (20.4%), 796~876 bp (40.7%) and 877~957 bp(22.2%) due to variable numbers of tandem repeats present within the gene. In this study it will be anticipated that this study can contribute to establish efficiently the countermeasure about the prevention and treatment of antibiotic bacterial infections.

This study was performed to compared the effectiveness of individual treatments (electrolyzed water: EW, organic acid, and ultrasound) and their combination on reducing foodborne pathogens from perilla leaves. Perilla leaves were innoculated with a cocktail of Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus. Inoculated perilla leaves were treated with EW combined with different concentration of acetic acid (0.5%, 1.0%, 1.5%, 2.0%) for 1 min at room temperature. Treatment of 3 pathogens on perilla leaves with electrolyzed water combined with ultrasound (25 kHz) and 0.5% acetic acid was also performed for 1 min. While the numbers of S. Typhimurium and B. cereus showed reduced with increasing acetic acid concentration, there is no difference in the number of S. aureus treated with EW containing 0.5% to 1.5% acetic acid. Discoloration was observed the perilla leaves treated with EW combined with more than 1.0% acetic acid. For all three pathogens, the combined treatment of EW and ultrasound resulted in additional 0.42 to 0.72 log10 CFU/g. The maxium reductions of S. Typhimurium and B. cereus were 0.95, 1.23 log10 CFU/g after treatment with EW combined with 0.5% acetic acid and ultrasound simultaneously. The results suggest that the treatment of EW combined with 0.5% acetic acid and ultrasound increased pathogens reduction compared to individual treatment.

Five kinds of selective media, such as mannitol salt agar (MSA), Baird-Parker agar (BPA), Baird- Parker supplemented with rabbit plasma fibrinogen (BPA+RPF), CHROMagar Staphylococcus aureus (CSA), and Petrifilm Staph Express count system (Petrifilm), were compared to recommend the optimum selective media for isolation of Staphylococcus aureus from agricultural products. Seventy four target and non target bacteria were inoculated on five selective media to analyze sensitivity and specificity. In the recovery test of injured S. aureus cells, S. aureus was exposed to acid (1% lactic acid for 10 min), heat (60oC for 90s), and cold (−20oC for 1h) conditions. And artificially contaminated agricultural products (iceberg lettuce, green pepper, and cherry tomato) was enumerated on five selective media. The sensitivity of BPA+RPF, CSA, Petrifilm, MSA, and BPA were 100%, 100%, 100%, 90.5%, 90.5%, respectively. In addition, the specificity of BPA+RPF, CSA, MSA, BPA and Petrifilm were 100%, 100%, 84.6%, 75.0%, 67.3%, respectively. However, no difference among five selective media was observed in recovery on injured S. aureus cell and enumeration from agricultural products. This results suggest that BPA+RPF and CSA are the optimum media for detection of S. aureus from agricultural products.

To determine current rate of antimicrobial resistance, a total of 236 isolates from milk samples of dairy cattle with mastitis in Korea during 2010-2011 were examined against 12 antimicrobials using disc diffusion method: 67 Staphylococcus aureus, 74 coagulase-negative Staphylococcus spp. (CNS), and 95 Escherichia coli isolates. The isolates examined in this study were submitted by Local Veterinary Service Laboratories located in 13 provinces and metropolitan cities nationwide. The highest rates of resistance among S. aureus isolates were against ampicillin (56.7%) and penicillin (56.7%), followed by kanamycin (11.9%). All S. aureus isolates were sensitive to lincomycin, amikacin, and cephalothin. Only one isolate showed resistance to tetracycline and oxacillin, respectively. Less than 10% of the S. aureus isolates presented resistance to erythromycin, neomycin, and gentamicin. Among CNS isolates, the most frequently observed resistance was to lincomycin (44.5%), followed by penicillin (28.3%), ampicillin (18.9%), tetracycline (17.5%), kanamycin (13.5%), and erythromycin (9.4%). All or most of the CNS isolates were sensitive to cephalothin, amikacin, neomycin, and gentamicin. The highest rate of resistance among E. coli isolates was against tetracycline (26.3%), followed by streptomycin (21%), neomycin (15%), kanamycin (12.6%), and gentamicin (10.5%). Amikacin was the only antimicrobial to which no E. coli isolates showed resistance. Around 10% of the S. aureus isolates and 15% of the CNS isolates showed resistance against three or more antimicrobials simultaneously, while more than 30% of the E. coli isolates did.

In this study, the contamination levels of total aerobic bacteria, E. coli, total coliform and S. aureus of seasoned dried fishes (SDF) in Korea were investigated. A total of 81 SDF samples were purchased randomly from 28 stores. Contamination range of total aerobic bacteria, total coliform and S. aureus were 150~1,700,000, 10~31,000and 10~220 CFU/g, respectively. E. coli was detected in only one samples in the qualitative test. We have analyzed quantitatively Staphylococcal enterotoxins (SE-type A, C and D) produced by S. aureus contaminated in SDF using a TECRA kit and standard curve. The curve equation was Y = 0.1499 * X + 0.1183 and maximum amount of SEs in SDF was 0.71 ng/ml. Reduction speed of S. aureus in SDF stored at 37 ℃ was the highest among the samples stored for 8 days at different temperature of 7, 18 and 37℃ . On the basis of the results, SDF in Korea can be contaminated by a variety of pathogenic bacteria. Therefore, precautionary measures are necessary for consumer protection, including the improvement of sanitary conditions in the processing plants in Korea.

The aim of this study was to evaluate the TEMPOⓇ STA automated most probable number (MPN) system for the enumeration of Staphylococcus aureus (S. aureus) in comparison with a standard culture method. Artificially inoculated food products with S. aureus - triangle kimbap, sliced spring onion, dried filefish fillet, danpatjuk (sweet red-bean porridge with small rice dumplings)- were tested in this study. Twenty-five grams of each of food samples were added into 225 ㎖ of sterilized phosphate buffered saline in a TEMPOⓇ stomacher bag followed by stomaching for 2 min. One milliliter of the stomached sample was added to a bottle of culture medium. Cards were filled and sealed in the automated filler and then were incubated for 24 h at 37℃. After incubation, the cards were placed in the automated TEMPOⓇ reader and MPN results were generated. For comparison with a culture method, decimal dilutions were prepared from the same homogenized samples described above, transferred onto Baird Parker and Baird Parker-Rabbit Plasma Fibrinogen (BP-RPF) agar plates, and then incubated at 37℃ for 24 h. The performance of TEMPOⓇ STA method is equivalent to the culture method using Baird Parker or BP-RPF agar count plate for the enumeration of S. aureus in foods, eliminating a time-consuming and laborious process.

Staphylococcus aureus is an important food-borne pathogen, which is present on the skin and mucosa of animals. Some of the S. aureus strains are causative agent of food poisoning syndrome. The aim of this study was to investigate the antimicrobial resistance of S. aureus isolates from raw meats in slaughterhouses during 2010. From 17,874 raw meat samples tested, a total of 190 S. aureus were isolated, for which antimicrobial susceptibility to 17 agents was examined using broth dilution method. Among isolates from beef, chicken and pork, 20 (51.3%), 20 (24.7%) and 9 (12.9%) were sensitive to all antimicrobials tested, respectively. Isolates from pork and chicken meats showed much higher resistance, compared to isolates from beef. Penicillin resistance was the most frequent among isolates from beef (35.3%) and pork (75.7%), while tetracycline resistance was among those from chicken meats (48.1%). A total of 3 methicillin resistant S. aureus (MRSA) were detected from beef (5.1%, 2/39) and pork (1.4%, 1/70). Although the prevalence of MRSA was low, the presence of antimicrobial resistant S. aureus such as MRSA suggests that further investigation and strict surveillance on MRSA and antimicrobial resistance are needed.

In this study, two duplex real-time PCR approach with melting curve analysis is presented for the detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus, which are important food-borne bacterial pathogens usually present in fresh and/or minimally processed vegetables. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the β-glucuronidase (uidA, E. coli), thermonuclease (nuc, S. aureus), hemolycin (hly, L. monocytogenes) and tetrathionate reductase (ttr, Salmonella spp.) genes. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the four pathogens; 80.6 ± 0.9 ℃,86.9 ± 0.5 ℃, 80.4 ± 0.6 ℃ and 88.1 ± 0.11 ℃ for S. aureus, E. coli O157:H7, L. monocytogenes and Salmonella spp.,respectively. For all the pathogens, the two duplex, real-time PCR was equally sensitive to uniplex real-time PCR,using same amounts of purified DNA, and allowed detection of 10 genome equivalents. When our established duplex real-time PCR assay was applied to artificially inoculated fresh lettuce, the detection limit was 10³ CFU/g for each of these pathogens without enrichment. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.