• 한국식품위생안전성학회지
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• 제34권6호
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• pp.557-561
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• 2019

본 연구에서는 가축분퇴비에 존재할 수 있는 식중독균의 검출을 위하여 기존의 배양을 이용한 방법을 대체할 수 있는 real-time PCR을 적용하고자 하였으며, 이에 따라 유전자 증폭에 영향을 미치는 DNA 추출 방법에 따른 식중독균 검출 효율을 비교하였다. 적용한 방법은 가열 처리, 유기용매 및 흡착제 처리, 효소 처리의 3가지로 구분할 수 있으며, 각 방법에 따른 DNA의 검출 효율을 실험 결과로 나타내었다. 가열 처리 방법에서는 가열 시간의 증가에 따라 DNA 검출 효율이 높아지는 경향을 나타냈으며, 유기용매 및 흡착제는 효과를 나타내지 않았고, 효소 처리의 경우에는 그람 양성균 보다는 그람 음성균의 DNA가 추출 효율이 더 높은 것으로 나타났다. 결론적으로 퇴비에서 30분 이상의 가열 처리와 효소의 처리를 통한 DNA 추출 방법은 real-time PCR을 적용한 식중독균 검출에 적합한 것으로 판단된다.

• 산업식품공학
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• 제21권3호
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• pp.191-200
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• 2017

There have been great efforts to develop a rapid and sensitive detection method to monitor the presence of pathogenic bacteria in food. While a number of methods have been reported for bacterial detection with a detection limit to a single digit, most of them are suitable only for the bacteria in pure culture or buffered solution. On the other hand, foods are composed of highly complicated matrices containing carbohydrate, fat, protein, fibers, and many other components whose composition varies from one food to the other. Furthermore, many components in food interfere with the downstream detection process, which significantly affect the sensitivity and selectivity of the detection. Therefore, isolating and concentrating the target pathogenic bacteria from food matrices are of importance to enhance the detection power of the system. The present review provides an introduction to the representative sample preparation strategies to isolate target pathogenic bacteria from food sample. We further describe the nucleic acid-based detection methods, such as PCR, real-time PCR, NASBA, RCA, LCR, and LAMP. Nucleic acid-based methods are by far the most sensitive and effective for the detection of a low number of target pathogens whose performance is greatly improved by combining with the sample preparation methods.

• 센서학회지
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• 제17권3호
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• pp.195-202
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• 2008

A biosensor based on the measurement of capacitance changes has been designed and fabricated for simple and realtime detection of bacteria. Compared to an impedance measurement technique, the capacitance measurement can make additional measurement circuits simpler, which improves a compatability for integration between the sensor and circuit. The fabricated sensor was characterized by detecting Escherichia coli(E. coli). The capacitance changes measured by the sensor were proportional to E. coli cell density, and the proposed sensor could detect $1{\times}10^6$ cfu/ml E. coli at least. The real-time detection was verified by measuring the capacitance every 20 minutes. After 7 hours of E. coli growth experiment, the capacitance of the sensor in the micro volume well with $4.5{\times}10^5$ cfu/ml of initial E. coli density increased by 20 pF, and that in another wells with $1.5{\times}10^6$ cfu/ml and $8.5{\times}10^7$ cfu/ml initial E. coli density increased by 56 pF and 71 pF, respectively. The proposed sensor has a possibility of the real-time detection for bacterial growth, and can detect E. coli cells with $1.8{\times}10^5$ cfu in nutrient broth in 5 hours.

• 미생물학회지
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• 제49권3호
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• pp.292-296
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• 2013

본 연구는 multiplex real-time PCR을 이용하여 Actinobacillus actinomycetemcomitans, Campylobacter rectus, Porphyromonas gingivalis, Tannerella forsythus, Treponema denticola, Prevotella intermedia 등과 같은 6종의 주요 치주 질환 원인 미생물들을 동시에 검출할 수 있는 분석 방법에 관한 것이다. 4개의 형광 염료를 사용하여 internal control과 함께 3개의 균종씩 나누어 분석하였으며, 분석 대상 균종 간 그리고 다른 종류의 구강 미생물 균종과의 간섭과 교차 반응이 없음을 확인하였다. 본 연구의 multiplex real-time PCR은 타액과 플라그 등의 다양한 샘플에 포함되어 있는 각 미생물들을 정성, 정량적으로 분석할 수 있었으며, 치주염 환자와 건강한 사람들에 대한 비교 분석 결과 분명한 차이를 발견 할 수 있었다.

• 한국군사과학기술학회지
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• 제22권4호
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• pp.575-580
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• 2019

In biological warfare, it is important to identify biological agents for proper treatment. We focused on developing a real-time RT-PCR kit that can detect multiple species of biological agents. AccuPower(R) Biothreat Real-Time RT-PCR Kit(v3.0) could detect Bacillus anthracis, Yersinia pestis, Vibrio cholerae, Francisella tularensis, Salmonella typhi, Rickettsia prowazekii, Variola virus, Hantaan virus, Yellow fever virus, Brucella spp., Shigella dysenteriae in a single reaction. The results showed that the kit was verified to be able to detect at least 0.005 ng of nucleotide and 10,000 CFU/ml of bacteria. Therefore, the kit is expected to be used as a rapid and sensitive detection kit for 11 species of biological agents within 2 hours.

• 한국축산식품학회지
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• 제31권6호
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• pp.851-857
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• 2011

This study was conducted to develop a real time reverse transcriptase-PCR (RT-PCR) method for the detection of viable Enterobacteriaceae in milk using primers based on the genes of ribosomal proteins S11 and S13 and to determine effects of heating and subsequent treatments on the threshold cycle (Ct) of the real time RT-PCR. Total RNA was isolated from 17 strains of bacteria including 11 strains of Enterobacteriaceae suspended in milk using a modified Tri reagent method. SYBR Green Master Mix was added to the RNA and the mixture was subjected to the real time RT-PCR. The Cts of eleven type strains of the Enterobacteriaceae in milk ($10^7$ cells) in the real time RT-PCR ranged from 21.5 to 24.6. However, the Cts of Pseudomonas fluorescens, Acinetobacter calcoaceticus, and three gram-positive bacteria were more than 40. The real time RT-PCR detected as low as $10^3$ cells in agarose gel electrophoresis. The Cts increased from 22.0 to 34.2 when milk samples contaminated with Escherichia coli ($10^7$ cells/mL) were heated at $65^{\circ}C$ for 30 min. In addition, subsequent incubation at $37^{\circ}C$ for 6 and 24 h increased the Cts further up to 36.2 and 37.2, respectively. Addition of RNase A to the bacterial suspension obtained from the heated milk and subsequent incubation at $37^{\circ}C$ for 1 h increased the Cts to more than 40. The results of this study suggests that pretreatment of bacterial cells heated in milk with RNase A before RNA extraction might enhance the ability to differentiate between viable and dead bacteria using real time RT-PCR.

• 한국환경보건학회지
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• 제42권3호
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• pp.189-195
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• 2016

Objectives: To ensure the microbiological safety of groundwater, it was confirmed whether waterborne pathogenic bacteria in groundwater samples tested positive for total coliforms in the Chungcheongnam-do Province region. Methods: Total colony counts, total coliforms and fecal coliforms were tested according to the process mandated by the drinking water quality testing standards of Korea. DNA was extracted from the samples, tested positive for total coliforms, and then subjected to real-time PCR to detect waterborne pathogenic bacteria. Results: A total of 115 samples were inadequate for drinking water. Thirty-one cases (27%) showed positive for fecal coliforms and nine cases (7.8%) showed total colony counts exceeding drinking water standards. Twenty-seven cases (23.5%) showed three items (total colony counts, total coliforms and fecal coliforms). Using the real-time PCR method, waterborne pathogens were detected in 57 cases (49.6%) in 115 samples. Seventy-eight cases of waterborne pathogenic bacteria were detected (including duplications): 27 cases of pathogenic E. coli (EPEC (19), ETEC (5), EHEC (1), EAEC (1) and EIEC (1)); 45 of Bacillus cereus; two of Yersinia spp.; two of Salmonella spp.; one of Staphylococcus aureus; one of Clostridium perfringens. Conclusion: The real-time PCR method can offer rapid and accurate detection of waterborne pathogenic bacteria. Therefore, this assay could be an alternative to conventional culture methods and can further ensure the microbiological safety of groundwater.

• 센서학회지
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• 제27권4호
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• pp.237-241
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• 2018

The real-time enrichment and detection of pathogens are serious issues and rapidly evolving field of research because of the ability of these pathogens to cause infectious diseases. In general, bacterial detection is accomplished by conventional colony counting or by polymerase chain reaction (PCR) after DNA extraction. As colony counting requires considerable time to cultivate, PCR is an attractive method for rapid detection. A small number of pathogens can cause diseases. Hence, a pretreatment process, such as enrichment is essential for detecting bacteria in an actual environment. Thus, in this study, we developed a microfluidic chip capable of performing rapid enrichment of bacteria and the extraction of their genes. A lectin, i.e., Concanavalin A (ConA), which shows binding affinity to the surface of most bacteria, was coated on the surface of magnetic particles to nonspecifically capture bacteria. It was subsequently concentrated through magnetic forces in a microfluidic channel. To lyse the captured bacteria, magnetic particles were irradiated by a wavelength of 532nm. The photo-thermal effect on the particles was sufficient for extracting DNA, which was consequently utilized for the identification of bacteria. Our device will help monitor the existence of bacteria in various environmental situations such as water, air, and soil.

• 한국치위생학회지
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• 제21권5호
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• pp.555-565
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• 2021

Objectives: This study was conducted to analyze peri-implantitis bacteria and identify their associations with health status and health activities. Methods: Gingival sulcus fluid at the implant's periodontal pockets sampled from the participants were analyzed by multiplex real time PCR. Results: Participants had strains in the order of 100% F. nucleatum, 98.0% E. corrodens, and 96.0% P. micra, and the correlation between C. rectus and E. nodatum was high (p<0.01). Diabetic group (P. gingivalis, P. nigrescens) hypertension (P. nigrescens), group with four or more periodontal pockets (P. gingivalis, T. dentica, P. intermedia, E. nodatum, and C. rectum), smoking (P. micra, E. corrodens), drinking (T. dentola), and scaling groups (C. rectus) were found to have more strains (p<0.05). Conclusions: Representative pathogenic microorganisms detected in periodontal pockets of implants were similar to dental periodontal pockets; however there were differences in the amount and distribution of microorganisms, and they were affected by health status and health behavior.

• International Journal of Oral Biology
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• 제41권3호
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• pp.149-154
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• 2016

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase ${\beta}-subunit$ gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC $33478^T$. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..