소아에서 폐렴구균 집락률 측정을 위해 비인두 흡인 물의 총 RNA를 이용한 실시간 중합효소 연쇄반응법

김영광 ( Young Kwang Kim ),이경훈 ( Kyoung Hoon Lee ),윤기욱 ( Ki Wook Yun ),이미경 ( Mi Kyung Lee ),임인석 ( In Seok Lim ) 대한소아감염학회 2016 Pediatric Infection and Vaccine Vol.23 No.3

목적: 폐렴구균은 주요 비인두 상재균으로, 주위 조직을 침범하여 침습성 감염을 일으킬 수 있어 보균 율에 대한 감시가 중요하다. 본 연구에서는 임상에서 비인두 흡인물로부터 추출하고 남은 RNA를 이용 하여 폐렴구균을 확인할 수 있는 실시간 중합효소 연쇄반응(real-time reverse transcription poly-merase chain reaction [RT-PCR])법을 구축하고, 보균율 측정에 있어서의 정확성과 이점을 확인하고자 하였다. 방법: 2014년 9월부터 10월까지 중앙대학교병원에 입원하여 호흡기 바이러스 RT-PCR 검사를 시행 받은 18세 이하의 소아들로부터 비인두 흡인물을 채취하였다. 먼저 배양법과 genomic DNA (gDNA) 를 이용한 real-time PCR을 시행하여 폐렴구균 검출률의 정확성을 확인하였다. 이 중 처음 20개의 검체 를 이용하여, 고전적인 배양법과 gDNA를 이용한 real-time PCR, 그리고 RNA를 이용한 real-time RT-PCR법을 시행하고 이를 비교 분석하였다. 결과: 총 157개의 검체에서 시행한 real-time PCR 검사는 기존의 배양검사와 일치율이 0.922 (P<0.01, Fisher exact test)로 매우 높았다. 배양검사에서 음성인 133개의 검체는 real-time PCR에서도 모두 음성을 보였다. 24개의 배양 양성 검체 중 21개의 검체는 real-time PCR에서도 양성이었지만, 나머지 검체는 음성 결과를 보였다. 20개의 검체에서 시행한 real-time RT-PCR 검사는 1개 검체를 제외 하고 배양법 및 real-time PCR과 결과가 일치하였다. 한편, 배양법을 시행하고 결과를 확인하기까지 는 총 26.5시간, real-time RT-PCR 검사에는 총 4.5시간이 소요되었다. 결론: 본 연구는 비인두 집락균 확인을 위한 real-time RT-PCR법의 확립과, 폐렴구균 보균율 측정에 있어서의 real-time RT-PCR 검사의 정확성 및 편의성을 보여주었다. Real-time RT-PCR 검사법 은 주요 세균들의 보균율 연구에 있어서 시간과 노력을 줄일 수 있는 좋은 방법이며, 폐렴구균의 역학 자료 수집에 큰 도움이 될 것으로 기대한다. Purpose: Monitoring pneumococcal carriage rates is important. We developed and evaluated the accuracy of a real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for the detection of Streptococcus pneumoniae . Methods: In October 2014, 157 nasopharyngeal aspirates were collected from patients aged <18 years admitted to Chung-Ang University Hospital. We developed and evaluated a real-time PCR method for detecting S. pneumoniae by comparing culture findings with the results of the real-time PCR using genomic DNA (gDNA). Of 157 samples, 20 specimens were analyzed in order to compare the results of cultures, real-time PCR, and real-time RT-PCR. Results: The concordance rate between culture findings and the results of real-time PCR was 0.922 (P <0.01, Fisher exact test). The 133 culture-negative samples were confirmed to be negative for S. pneumoniae using real-time PCR. Of the remaining 24 culture-positive samples, 21 were identified as S. pneumonia -positive using real-time PCR. The results of real-time RT-PCR and real-time PCR from 20 specimens were consistent with culture findings for all S. pneumoniae -positive samples except one. Culture and real-time RT-PCR required 26.5 and 4.5 hours to perform, respectively. Conclusions: This study established a real-time RT-PCR method for the detection of pneumococcal carriage in the nasopharynx. Real-time RT-PCR is an accurate, convenient, and time-saving method; therefore, it may be useful for collecting epidemiologic data regarding pneumococcal carriage in children.

즉석 섭취 식품에서 Staphylococcus aureus와 Bacillus cereus 검출을 위한 Conventional PCR과 Real-Time PCR법 비교

이유시,김석환,김미경,김순한 한국식품위생안전성학회 2021 한국식품위생안전성학회지 Vol.36 No.2

Staphylococcus aureus와 Bacillus cereus는 식중독을 일으키는 주요한 원인균 중 하나로 각종 식품에서 검출되면서 많은 주의가 요구되고 있다. 식중독 발생의 원인균을 신속하고 정확하게 검출할 수 있는 여러 가지 방법 중 DNA 분석을 기반으로 하는 PCR 검출법이 보편적으로 사용되고 있다. 본 연구에서는 샌드위치와 같은 즉석 섭취 식품에서 식중독을 유발할 수 있는 S. aureus와 B. cereus 의 신속검출을 위해 conventional PCR과 real-time PCR의 특이도 및 민감도를 비교하였다. 그 결과, 검출한계 범위가 배양액에서는 cPCR의 경우 S. aureus (104-106 CFU/mL), B. cereus (103-105 CFU/mL) 이였고, real-time PCR의 경우 S. aureus (103-106 CFU/mL), B. cereus (102-105 CFU/mL) 이였다. 식품에서는 cPCR의 경우 S. aureus (104-106 CFU/mL), B. cereus (103-105 CFU/mL) 이고, real-time PCR의 경우 S. aureus (104-106 CFU/mL), B. cereus (103-105 CFU/mL) 으로 나타났다. Real-time PCR법이 cPCR법 보다 10배 이상 더 민감한 것으로 나타났다. 따라서 real-time PCR은 우수한 민감도를 지닌 검출기법으로 식중독 세균 검출에 있어 매우 효과적인 방법으로 사료된다. In the present study, the performance of different PCR assays was compared for the rapid detection of S. aureus and B. cereus in ready-to-eat foods such as sandwiches. The limit of detection (LOD) and specificity of two assays (conventional PCR and real-time PCR) were compared. The LOD of real-time PCR ranged from 103 to 106 CFU/mL for S. aureus and from 102 to 105 CFU/mL for B. cereus. The LOD of conventional PCR ranged from 104 to 106 CFU/mL for S. aureus and from 103 to 105 CFU/mL for B. cereus. The LOD of real-time PCR assay for S. aureus and B. cereus in sandwiches ranged from 104 to 106 CFU/mL and from 103 to 105 CFU/mL, while the LOD of conventional PCR was from 104 to 106 CFU/mL and from 103 to 105 CFU/mL, respectively. The LOD of the real-time PCR was 10-fold higher than that of the conventional PCR. In conclusion, these results show that real-time PCR could be used as an effective screening test for the rapid detection of S. aureus and B. cereus in sandwiches compared to conventional PCR.

Architecture of Task Manager for Real Time OS Explaining Real Time Operating Systems Issues

Javed Ahmad Shaheen MSCS 보안공학연구지원센터 2016 International Journal of Grid and Distributed Comp Vol.9 No.9

Complications of embedded applications are increasing. Within due time delivery to the market is also a pressure. So, the demand and use of real time operating systems is also increasing. However, it is an redundant fact that the Real Time Operating System (Real Time OS) can significantly degrade the performance. To face performance degradation, a Real-Time Task Manger (RTTM) has been presented in this paper. The Real-Time Task Manger is a hardware based extension to the processor that decreases performance bottleneck attributed to Real Time Operating System. Real-Time Task Manger decreases these performance bottlenecks introduced by Real Time Operating System by its hardware based architecture. The architecture of Real-Time Task Manger is being discussed in this paper provides an aid to deal with some common operations of Real Time Operating System that reason the performance degradation. For example event management, time management, and task scheduling. These operations have a property of some inborn parallelism. The Real-Time Task Manger uses this property to complete these operations in a constant time, and as a result, minimizes the overhead introduced by Real Time Operating System. The proposed architecture yields two benefits: It reduces the processor time taken by Real Time Operating System, and improves the response time to a considerable amount.

실시간 임베디드 리눅스에서 다양한 주기적 타스크의 실시간 메커니즘 성능 분석

고재환,최병욱 한국로봇학회 2012 로봇학회 논문지 Vol.7 No.4

It is a real-time system that the system correctness depends not only on the correctness of the logical result of the computation but also on the result delivery time. Real-time Operating System (RTOS) is a software that manages the time of a microprocessor to ensure that the most important code runs first so that it is a good building block to design the real-time system. The real-time performance is achieved by using real-time mechanisms through data communication and synchronization of inter-task communication (ITC) between tasks. Therefore, test on the response time of real-time mechanisms is a good measure to predict the performance of real-time systems. This paper aims to analysis the response characteristics of real-time mechanisms in kernel space for real-time embedded Linux: RTAI and Xenomai. The performance evaluations of real-time mechanism depending on the changes of task periods are conducted. Test metrics are jitter of periodic tasks and response time of real-time mechanisms including semaphore, real-time FIFO, Mailbox and Message queue. The periodicity of tasks is relatively consistent for Xenomai but RTAI reveals smaller jitter as an average result. As for real-time mechanisms, semaphore and message transfer mechanism of Xenomai has a superior response to estimate deterministic real-time task execution. But real-time FIFO in RTAI shows faster response. The results are promising to estimate deterministic real-time task execution in implementing real-time systems using real-time embedded Linux.

실시간-염기순서기반증폭(Real-Time Nucleic Acid Sequence-based Amplification)을 통한 장바이러스 검출

박경운,전선희,성기형,송상훈,김홍빈,송정한,최은화,박성섭,김의종 대한임상미생물학회 2010 Annals of clinical microbiology Vol.13 No.2

Background: Enteroviruses are the most frequent etiologic agents of aseptic meningitis and are estimated to be the cause of 70% to 90% of viral meningitis cases. Enterovirus diagnosis can be difficult because clinical features vary according to patient immunity and age. The purpose of this study was to evaluate the performance of the real-time nucleic acid sequence-based amplification (NASBA) assay compared to that of the real-time nested RT-PCR assay for enterovirus detection. Methods: This study was performed on 96 patients suspected of aseptic meningitis based on clinical features. RNA was extracted using NucliSENS EasyMAG and real-time NASBA assay was performed using NucliSENS EasyQ Enterovirus and NucliSENS EasyQ Basic 2. We also executed in-house real-time nested RT-PCR assay for RNA extracted via QIAamp Viral RNA Mini. Results: The positive rate of real-time NASBA assay was 45.8% for enterovirus detection. The positive rate of first real-time reverse transcription PCR was 22.9% and the second real-time PCR was 57.3%. The concordant rate of the real-time NASBA assay and first real-time reverse transcription PCR was 75.0%. The concordant rate of the real-time NASBA assay and second real-time PCR was 86.5%. Conclusion: The detection of enteroviruses using the real-time NASBA assay is less prone to cross-contamination and is simple, without the need for reverse transcription. We conclude that the NASBA assay is an effective method for the rapid diagnosis of aseptic meningitis. (Korean J Clin Microbiol 2010;13:53-58)

분말식품에서 Cronobacter spp. 검출을 위한 Real-Time PCR과 배지배양법의 비교검증

천정환,송광영,김선영,현지연,김윤경,황인균,곽효선,서건호,Chon, Jung-Whan,Song, Kwang-Young,Kim, Sun-Young,Hyeon, Ji-Yeon,Kim, Yun-Gyeong,Hwang, In-Gyun,Kwak, Hyo-Sun,Seo, Kun-Ho 한국미생물학회 2011 미생물학회지 Vol.47 No.1

본 연구에서는 분말 식품에서 real-time PCR과 배지배양법을 사용하여 Cronobacter spp.를 검출하는 방법이 비교검증 되었다. 조제분유, 이유식, 미숫가루에 Cronobacter를 인위적으로 접종시킨 후, 식품공전의 방법에 따라 멸균증류수와 Enterobacteriaceae enrichment (EE) broth에서 각각 1, 2차 증균배양 하였으며, Druggan-Forsythe-Iversen에 선택배양하여 Cronobacter를 검출하였다. Real-time PCR은 멸균증류수 및 EE broth에서 1 ml을 채취한 후 DNA를 추출하여 시행하였다. 실험결과 모든 식품에서 배지배양법과 real-time PCR간에는 통계학적 유의차가 존재하지 않았다(p>0.05). 한편 모든 실험회차에서 real-time PCR 수행 시, 1차 증균액인 멸균증류수에서의 양성검출율이 2차 증균액인 EE broth에서보다 높았는데, 이는 2차 증균액 내의 구성성분 중 일부분이 real-time PCR의 반응을 저해했기 때문으로 사료된다. 연구결과를 종합해 볼 때, 1차 증균 후, real-time PCR을 통해 Cronobacter를 검출하는 방법은 정확한 민감도를 보이면서도 시간과 노동력을 절감할 수 있는 효과적인 방법으로 사료된다. The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

Propidium monoazide와 real-time PCR을 이용한 살아있는 Enterococcus faecalis의 선택적인 검출

김신영,이승종,김의성,서덕규,송윤정,정일영 大韓齒科保存學會 2008 Restorative Dentistry & Endodontics Vol.33 No.6

세균의 검출에 있어서 polymerase chain reaction (PCR) 방법은 기존의 plate counting과 달리 빠르게 세균을 검출할 수 있다. 하지만 세균이 죽은 후에도 DNA는 장기간 존재할 수 있기 때문에, DNA에 기초한 분석은 살아있는 세균과 죽은 세균을 구분할 수 없다. 최근에 DNA extraction전에 propidium monoazide (PMA)를 처리하여 살아있는 세균만 선택적으로 검출하는 방법이 제시되었다. PMA는 손상된 세포막만 통과하여 죽은 세포의 DNA와 빛 노출 하에서 결합하여 PCR이 증폭되는 것을 막는다. Enterococcus faecalis는 근관치료의 실패에 있어서 중요한 원인이 되는 세균으로 제시되어 왔다. 그리고 Chlorhexi-dine (CHX)은 E. fecalis의 제거에 있어서 효과적인 약제임이 밝혀졌다. 이번 실험의 목적은 세균 수의 측정에 있어서, PMA 처리와 real-time PCR 방법의 적용 가능성을 기존의 plate counting과 비교하여 알아보는 것이다. 또한 E. faecalis에 대한 2% CHX의 살균 효과를 PMA 처리 후 real-time PCR 방법을 사용하여 알아보는 것이다. 실험 방법으로 먼저 살아있는 세균과 죽은 세균을 다른 비율로 섞어서 PMA를 처리한 후 real-time PCR을 시행하여 PMA가 빛 노출 하에서 죽은 세균의 DNA와 결합하는 효과를 나타내는지 알아보았다. 다음으로 PMA처리 후 real-time PCR 방법을 이용하여 살아있는 세균의 양을 측정한 것을 plate counting으로 얻은 CFU와 비교하였다. 마지막으로 2% CHX의 처리시간을 다르게 하였을 때 E. faecalis에 대한 살균 효과를 PMA 처리 후 real-time PCR방법을 사용하여 알아보았다. 실험 결과로 살아있는 E. faecalis의 비율이 감소할수록 Ct value는 증가하였다. 그리고 PMA 처리 후 real-time PCR방법을 이용하여 세균의 양을 측정한 것과 plate counting으로 얻은 CFU사이에는 Optical density (OD) 값이 1.0일 때까지는 상관관계가 있었다. 하지만 OD 값이 1.5일 때는, PMA를 처리한 후 real-time PCR을 시행했을 때 측정된 살아있는 세균의 양이 감소하였음에 반해서 plate counting에 의한 CFU는 계속 증가하였다. 마지막으로 2% CHX을 오래 적용할수록 살아있는 E. faecalis의 상대적인 양이 감소하는 것을 PMA 처리와 real-time PCR 방법을 이용해 확인하였다. Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treat-ment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

준실시간 품질처리 기법을 활용한 해수위 관측자료의 품질개선 방안

석민준,황창수,이수호,이준식,송동훈,박상표 한국수로학회 2023 한국수로학회지 Vol.12 No.2

립해양조사원(KHOA, Korea Hydrographic and Oceanographic Agency)은 국가해양관측망의 안정적인 운영과 해양관측자료의 품질 향상을 위하여 관측시설물 유지관리와 해양관측자료의 품질처리(실시간 및 비실시간)를 수행하고 있다. 실시간 품질처리는 다양한 물리적 알고리즘 및 기준을 이용하여 자동 품질처리를 수행하는 것으로, 실시간 품질처리가 완료된 관측자료는 ‘국립해양조사원 홈페이지 누리집’을 통해 제공 중이다. 비실시간 품질처리는 매월 초순에 전월의 관측자료를 대상으로 품질관리 담당자가 수동 품질처리를 수행하는 것이며, 비실시간 품질처리가 완료된 자료는 가공 및 분석을 통해 통계 자료, 1시간 조위 등을 산출하여 해당 정보들을 간행물에서 제공하고 있다. 본 연구는 국내 해양관측자료의 품질처리 방법 개선을 위하여 기존 실시간, 비실시간 품질처리 기법 외 적용이 가능한 준실시간 품질처리 기법을 조사하고, 해당 기법을 해수위 관측자료에 적용 및 실험하여 활용 가능성을 검토하였다. 해양관측자료 준실시간 품질처리 기법의 조사 결과, 다양한 해양과 관련된 국제적인 유관기관(IOC, GLOSS 등)에서는 해수위(조위) 관측자료의 품질개선을 위해 SELENE(SEa LEvel NEar-real time quality control processing) 알고리즘의 적용을 적극 권장하고 있다. 이에 국립해양조사원의 조위관측소에서 수집한 조위 자료에 대해 품질처리 최적화 실험을 통해 적합한 매개변수를 산출하였으며, 해당 기법을 적용하여 품질처리 정확도를 분석하였다. 분석 결과, 특이상황을 제외한 조위 관측자료의 준실시간 품질처리 정확도는 평균 90% 이상으로 매우 양호하게 나타났다. 따라서 실시간 및 비실시간 품질검사 외 해당 준실시간 품질처리 기법을 품질검사 현업에 도입·적용한다면, 조위 관측자료의 품질개선과 더불어 신속한 품질처리 자료 생산으로 해양정보 서비스 활용도 향상에 큰 도움이 될 것으로 생각된다. The main responsibility of Korea Hydrographic and Oceanographic Agency (KHOA) is to operate the Korea Ocean Observing and Forecasting System(KOOFS) and enhance the quality of observed oceanographic data. To achieve this, KHOA maintains facilities and quality control of observed oceanographic data in real-time and non-real time. real-time quality control means automatic quality control using various physical algorithms. The data for which real-time quality control has been completed is provided on the the KHOA Homepage. Non-real time quality control means manual processing by oceanographic experts.statistical data, hourly tidal levels, and other information, which is made available through publications. The purpose of study is to enhance the quality processing performance of marine observation data by applying near-real time quality control techniques in addition to the existing quality control methods. First, a survey was conducted on the various quality processing techniques used by various international organizations. And then, among the surveyed techniques, suitable methods were applied to sea level data for experimentation. According to the results of the investigation into near-real time quality management technology, various institutions strongly recommend the use of the SELENE(SEa LEvel NEar-real time quality control processing) algorithm for improving the quality of sea level data. To determine suitable parameters, optimization experiments were conducted using sea level data observed at KHOA's tidal observation station. This algorithm was then utilized to measure the accuracy of quality control data. As a result, the accuracy of near-real time quality-managed sea level data except in special situation was found to be above 90% on average. If it would use the near-real time quality management technique and utilizing it in conjunction with real-time and non-real time quality management would not only enhance the quality of sea level observation data but also enable swift quality management. Therefore, it is expected to significantly contribute to the improvement of marine information services.

Evaluation of Two Commercial HLA-B27 Real-Time PCR Kits

조은해,이상곤,석정호,박보야나,이은희 대한진단검사의학회 2009 Annals of Laboratory Medicine Vol.29 No.6

Background : The standard PCR with sequence-specific primers (SSP) is a widely used method of HLA-B27 typing in clinical practice. The aim of our study was to evaluate 2 Korean HLA-B27 kits with different real-time PCR chemistries. Methods : To validate the accuracy of real-time PCR kits, we selected 28 HLA-B27-positive samples and 33 HLA-B27-negative samples with a wide range of different HLA-B specificities typed by standard PCR-SSP. The 2 real-time PCR kits used were the AccuPower HLA-B27 real-time PCR kit (Bioneer, Korea) with TaqMan probes and the Real-QTM HLA-B*27 detection kit (BioSewoom, Korea) with SYBR Green I dye for melting curve analysis. Results : All 61 samples typed by PCR-SSP demonstrated a perfect concordance with the 2 realtime PCR assays. It was possible to clearly discriminate between HLA-B27-positive and -negative samples in both real-time assays. Conclusions : In summary, both real-time PCR assays for HLA-B27 were fast, reliable, well-adapted for routine laboratory testing, and attractive alternatives to the conventional PCR-SSP method. Background : The standard PCR with sequence-specific primers (SSP) is a widely used method of HLA-B27 typing in clinical practice. The aim of our study was to evaluate 2 Korean HLA-B27 kits with different real-time PCR chemistries. Methods : To validate the accuracy of real-time PCR kits, we selected 28 HLA-B27-positive samples and 33 HLA-B27-negative samples with a wide range of different HLA-B specificities typed by standard PCR-SSP. The 2 real-time PCR kits used were the AccuPower HLA-B27 real-time PCR kit (Bioneer, Korea) with TaqMan probes and the Real-QTM HLA-B*27 detection kit (BioSewoom, Korea) with SYBR Green I dye for melting curve analysis. Results : All 61 samples typed by PCR-SSP demonstrated a perfect concordance with the 2 realtime PCR assays. It was possible to clearly discriminate between HLA-B27-positive and -negative samples in both real-time assays. Conclusions : In summary, both real-time PCR assays for HLA-B27 were fast, reliable, well-adapted for routine laboratory testing, and attractive alternatives to the conventional PCR-SSP method.

Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce

Lee, Na-Ri,Kwon, Kyung-Yoon,Choi, Sung-Wook,Koo, Min-Seon,Chun, Hyang-Sook The Korean Society of Food Hygiene and Safety 2011 한국식품위생안전성학회지 Vol.26 No.2

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 ${\beta}$-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 $T_m$ values demonstrating the specific and efficient amplification of the four pathogens; $80.6{\pm}0.9^{\circ}C$, $86.9{\pm}0.5^{\circ}C$, $80.4{\pm}0.6^{\circ}C$ and $88.1{\pm}0.11^{\circ}C$ 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 $10^3$ 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. 본 연구에서는 신선편의 채소와 과일에서 주로 검출되는 대장균(E. coli Ol577:H7), 리스테리아(L. monocytogenes), 살모넬라(Salmonella, spp.), 형색포도상구균(S. aureus)을 검출하고 동정할 수 있는 real time PCR 방법을 melting curve 분석을 활용하여 single tube 반응으로 두 종의 식중독균을 동시 검출하는 방법을 개발하고자 하였다. 대장균의 ${\beta}$-glucuronidase (uidA), 황색포도상구균의 thermonuclease (nuc), 리스테리아의 hemolycin (hly), 살모넬라의 tetrahionate reductase (ttr) 를 특이적으로 검출할 수 있는 4종의 프라이머 세트에 대한 real-time PCR의 melting curve 분석을 통하여 황색포도상구균과 대장균 동시분석 시 $T_m$ 값이 $80.6{\pm}0.9^{\circ}C$와 $86.9{\pm}0.5^{\circ}C$, 리스테리아와 살모넬라 동시분석 시 Tm 값이 $80.4{\pm}0.6^{\circ}C$와 $88.1{\pm}0.11^{\circ}C$로 확인하였고, 그 결과 정제되어진 각 식중독균의 genomic DNA를 주형으로 한 duplex real-time PCR 방법이 uniplex real-time PCR 방법과 마찬가지로 10 genome equivalents 까지 검출할 수 있는 민감도를 나타내었다. 또한, 양배추에 네 종의 식중독균을 접종하고, 증균배양 없이 DNA를 추출하여 duplex real-time PCR 을 수행한 결과 모든 식종목균에서 $10^3$ CFU/g의 검출 한계를 나타내었다. 결과적으로 개발된 melting curve 분석을 이용한 duplex real-time PCR 방법은 식품안전 증진을 위한 시간, 노동력, 비용 절감에 있어서 유효한 방법이 될 것으로 판단된다.