PCR-DGGE를 이용한 유기농 채소의 유해 미생물 신속 검지

권오연,손석민 한국산업식품공학회 2011 산업 식품공학 Vol.15 No.4

현재 식중독 미생물의 검사에 전통적인 배지법이 다수를 차지하고 있으나 PCR이나 면역 분석법과 같은 신속 검출법들의 사용이 증가하는 추세이다. 그러나 대개 speciesspecific 프라이머를 이용한 PCR에 의한 DNA 증폭산물을 전기영동을 통해 확인하는 방법을 사용하고 있는데 이는 각종 균주에 대해 각기 다른 프라이머를 사용하여야하는 단점이 있다. 이러한 단점을 보완하기 위하여 본 연구에서는 여러 가지 균주를 동시에 신속하게 검지할 수 있도록 PCR-DGGE 기술을 연구하게 되었다. 그 결과 6종(S. typhimurium, P. fluorescens, B. cereus, S. aureus, E. coli, L. monocytogenes)의 유해 미생물을 선정하였고 DGGE 상에서 확실한 분리능을 보여 유해미생물의 인공마커로 사용할 수 있음을 보였다. 또한 실제 PCR-DGGE 기법을 사용하기 위하여 채소에서의 유해미생물 검출 감도를 확인한 결과 B. cereus를 제외한 5종(S. typhimurium, P. fluorescens, S. aureus, E. coli, L. monocytogenes)의 균들은 모두 101 CFU/g까지 검출할 수 있었고 B. cereus는 103 CFU/g이상 채소에 존재할 때 검출할 수 있었다. 또한 균질화 방법에 따라서 식물 DNA와 유해 미생물 간의 경쟁적 증폭현상이 일어남도 확인할 수 있었다. 이로써 본 연구에서는 유해 미생물의 검지, 인공마커의 제조, 식물 DNA와 유해미생물간의 경쟁적 증폭 현상 방지책과 유해미생물의 검출 감도 확인 및 염기서열을 통한 동정을 통해 PCR-DGGE가 식품에서 유해 미생물의 신속 검지 방법으로써 활용할 수 있을 것으로 사료된다. 향후 국내 식품소비 패턴이 유기농 채소의 수요와 공급이 날로 증대 되고 있기 때문에 PCR-DGGE 기법이 유기농 채소들의 식중독 균에 대한 database를 구축하여 안전성을 확보하는데 기여할 수 있다고 생각된다. In this study the polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens in fresh originally grown vegetables. A universal primer (341GCf/534r) was selected for its ability to amplify the V3 region of 16S-rRNA genes in their target pathogens (Salmonella typhimurium, Pseudomonas fluorescens, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, E. coli). The 194 bp fragments in PCR were successfully duplicated as expected. The amplified fragments of the same size from six different pathogens also showed good separation upon DGGE. The detection limit of PCR-DGGE for six pathogens in fresh-cut lettuces were over 105 CFU/g when sampled by stomaching. However, when the sampling method was changed from stomaching to shaking, the detection limit of six pathogens in organic vegetables was shown to increase by over 101 CFU/g, but only those of B. cereus were over 103 CFU/g. Therefore, PCR-DGGE was shown to be a reliable method for the detection of pathogens in fresh-cut vegetables.

Comparative Analysis of Detection Methods for Food-borne Pathogens in Fresh-cut Agricultural Materials

Hye-Jeong Jang(장혜정),Hye-Jeong Kim(김혜정),Ji-in Park(박지인),Sun-Nyoung Yu(유선녕),Bo-Bae Park(박보배),Gang-Ja Ha(하강자),Soon-Cheol Ahn(안순철),Dong-Seob Kim(김동섭) 한국생명과학회 2021 생명과학회지 Vol.31 No.1

건강에 대한 관심 증대와 1인 가구 증가라는 사회구조적인 변화로 이용하기 편리한 농산물에 대한 소비가 증가하고 있다. 대부분의 신선 농산물은 가열하지 않고 섭취하는 경우가 많기 때문에 식품 매개 병원체에 쉽게 노출될 수 있어 세계적으로 과채류가 원인인 식중독 사고의 보고가 증가하고 있다. 이에 본 연구에서는 신선 농산물의 미생물학적 품질을 평가하고 식중독균 검출 방법을 비교 분석하고자 하였다. 신선 농산물 중 채소류 129건을 구입하여 배양기반 방법으로 식중독균을 분석한 결과, non-pathogenic Escherichia coli (3.9%), Bacillus cereus (31.8%), Clostridium perfringens (5.4%), Yersinia enterocolitica (0.8%), enterohemorrhagic E. coli (0.8%)가 검출되었다. 이러한 식중독균의 분석에는 증균 배양과정이 중요하게 작용을 하며 균주의 순수 분리 및 확인 동정에까지 상대적으로 많은 시간과 노력이 요구된다. 따라서 증균 배양의 과정 없이 식중독균을 신속하게 검출 할 수 있는 PCRDGGE를 수행하여 배양 기반의 분석법과 비교하였다. 비병원성 대장균은 배양 기반 방법에서 검출되지 않았음에도 PCR-DGGE에서는 검출된 경우가 2건이 있었다. 본 연구에서 사용한 대장균 정량 분석방법은 시료를 10배 희석한 후 배양하는 과정에서 시료의 손실 가능성과 검출 한계가 높은 단점으로 PCR-DGGE가 균종의 확인에 더욱 용이할 것으로 보였다. 저위해성 식중독균은 배양 기반 방법보다 PCR-DGGE에서 검출 한계가 높은 것으로 보였다. 고위해성 식중독균은 배양 기반 방법보다 PCR-DGGE (10 CFU/g)에서 검출 한계가 낮아 균종 확인과 검출에 용이하다고 판단되었고 이를 통해 채소류에서 고위해성 식중독균의 잠재적 위험성을 확인하였다. 본 연구의 결과는 신선 농산물의 미생물 위해 평가와 기준 설정을 위한 기초 자료로 활용될 수 있으며 신선 농산물 관련 식중독균 검출 방법의 개선과 식중독 발생 예방에 기여할 것으로 기대한다. The consumption of fresh-cut agricultural materials is increasing due to increased public interest in health and the increase of single-person households. Most fresh-cut agricultural materials can be eaten without heating, thus easily exposing the consumer to food-borne pathogens. As a result, food-borne diseases are increasing worldwide. In the analysis of food-borne pathogens, it is important to detect the strains, but this is time consuming and laborious. Alternative detection methods that have been introduced, include polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), which is performed without prior culturing. Samples of fresh-cut agricultural materials, such as vegetables, were analyzed by the culture-based method. In 129 samples, non-pathogenic Escherichia coli (3.9%), Bacillus cereus (31.8%), Clostridium perfringens (5.4%), Yersinia enterocolitica (0.8%), and enterohemorrhagic E. coli (0.8%) were detected. Eight samples contaminated with bacteria were randomly selected, further analyzed by PCR-DGGE, and compared with the culture-based method. Two cases detected non-pathogenic E. coli by PCR-DGGE only, despite a lack of detection by the culture method. It was supposed there was possibility of sample loss during its 10-fold dilution for appropriate cultivation. In the detection of high-risk food-borne pathogens, it was found that the detection limit was lower in PCR-DGGE than in the culture-based method (10 CFU/g). This suggests that PCR-DGGE can be alternatively used to detect strains. On the other hand, low-risk food-borne pathogens seem to have higher detection limits in PCR-DGGE. Consequentially, this study contributes to the improvement of food-borne pathogen detection and the prevention of its related-diseases in fresh-cut agricultural materials.

PCR-DGGE 방법을 이용한 북한강 수계 호수의 플랑크톤 군집 분석

김윤정,김민경,이상돈 한국습지학회 2012 한국습지학회지 Vol.14 No.3

식물플랑크톤의 동정은 숙련된 전문가에게도 어려운 과제이다. 별 특징없는 외형과 다양한 크기와 종은 형태학적으로 구분하기에 어려움이 있다. 본 연구에서는 미생물 군집의 다양성을 분석하는데 효과적인 fingerprinting 기법인 PCR-DGGE 방법을 사용하여 이런 형태학적 동정의 제한점을 보완하고자 하는데 목적이 있다. 5곳의 호수 샘플로부터 2008년 8월 총 46개의 band를 찾을 수 있었고, 2008년 11월 총 26개 band를 찾 을 수 있었다. 이 fingerprint 결과는 각각 다른 샘플링 장소를 비교하는데 용이하였다. 본 연구에서 PCR-DGGE 방법은 북한강 호수들의 플랑크톤 군집의 다양성을 파악하는데 사용되었고, 이 DGGE 기법이 플랑크톤의 동정 기법으로써의 가능성을 검토해보았다.

PCR-DGGE를 이용한 유기농 채소의 유해 미생물 신속 검지

권오연 ( Oh Yeoun Kwon ),손석민 ( Seok Min Son ) 한국산업식품공학회 2011 산업 식품공학 Vol.15 No.4

In this study the polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens in fresh originally grown vegetables. A universal primer (341GCf/534r) was selected for its ability to amplify the V3 region of 16S-rRNA genes in their target pathogens (Salmonella typhimurium, Pseudomonas fluorescens, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, E. coli). The 194 bp fragments in PCR were successfully duplicated as expected. The amplified fragments of the same size from six different pathogens also showed good separation upon DGGE. The detection limit of PCR-DGGE for six pathogens in fresh-cut lettuces were over 105 CFU/g when sampled by stomaching. However, when the sampling method was changed from stomaching to shaking, the detection limit of six pathogens in organic vegetables was shown to increase by over 101 CFU/g, but only those of B. cereus were over 103 CFU/g. Therefore, PCR-DGGE was shown to be a reliable method for the detection of pathogens in fresh-cut vegetables.

Senior Thai Fecal Microbiota Comparison Between Vegetarians and Non-Vegetarians Using PCR-DGGE and Real-Time PCR

( Supatjaree Ruengsomwong ),( Yuki Korenori ),( Naoshige Sakamoto ),( Bhusita Wannissorn ),( Jiro Nakayama ),( And Sunee Nitisinpraser ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.8

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

Rapid Identification of Bifidobacteria in Dairy Products by Gene-targeted Species-specific PCR Technique and DGGE

Hong, Wei-Shung,Chen, Ming-Ju Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.12

In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.

황 산화를 통해 퍼클로레이트를 분해하는 미생물 군집 분석

김영화(Young-Hwa Kim),한경림(Kyoung-Rim Han),황희재(Heejae Hwang),권혁준(Hyukjun Kwon),김예림(Yerim Kim),김건우(Kwonwoo Kim),김희주(Heejoo Kim),손명화(Myunghwa Son),최영익(Young-Ik Choi ),성낙창(Nak-Chang Sung),안영희(Yeonghee Ahn) 한국생명과학회 2016 생명과학회지 Vol.26 No.1

퍼클로레이트(ClO₄<SUP>-</SUP>)는 지표수 및 토양/지하수에서 검출되는 신규 오염물이다. 이전 연구에서 저렴한 원소 황(elemental sulfur, S<SUP>0</SUP>) 입자와 쉽게 구할 수 있는 활성슬러지를 이용하여 독립영양방식으로 ClO₄<SUP>-</SUP>를 제거할 수 있다는 실험적 증거가 제시되었다. 또한 식종균으로서 농화배양 미생물을 사용했을 때 활성슬러지보다 제거효율과 시간면에서 우수한 결과를 나타내었다. 그래서 본 연구에서는 황을 산화하여 독립영양방식으로 ClO4-를 분해하는 농화배양 미생물 군집을 PCR-DGGE로 분석하였다. 이 농화배양 미생물은 초기농도가 약 120 mg ClO₄<SUP>-</SUP>/l 일 때 7일 후 99.71% 이상의 ClO₄<SUP>-</SUP> 제거 효율을 나타내었다. 농화배양 미생물과 그것의 식종균으로 부터 genomic DNA를 추출하여 16S rRNA 유전자의 PCR-DGGE 분석에 사용하였다. PCR-DGGE 분석결과 농화배양 미생물과 식종균 시료들이 다른 밴드패턴을 나타냄에 따라 이 두 시료의 군집조성이 다름을 확인하였다. 이는 농화배양되는 동안 식종된 미생물이 그 환경에 잘 생장하는 미생물로 군집조성이 변화한 것으로 여겨진다. 농화배양 미생물군집에는 β-Proteobacteria, Bacteroidetes, 그리고 Spirochaetes 강에 속하는 개체군들이 우점하는 것으로 나타났다. 향후 이 우점 개체군들의 순수분리와 더불어 황 산화를 통한 ClO₄<SUP>-</SUP> 분해 환경에서 이들의 대사적 역할을 규명할 필요가 있다. Perchlorate (ClO₄<SUP>-</SUP>) is an emerging pollutant detected in surface water, soil, and groundwater. Previous studies provided experimental evidence of autotrophic ClO₄<SUP>-</SUP> removal with elemental sulfur (S<SUP>0</SUP>) particles and activated sludge, which are inexpensive and easily available, respectively. In addition, ClO₄<SUP>-</SUP> removal efficiency was shown to increase when an enrichment culture was used as an inoculum instead of activated sludge. PCR-DGGE was employed in the present study to investigate the microbial community in the enrichment culture that removed ClO₄<SUP>-</SUP> autotrophically. Microorganisms in the enrichment culture showed 99.71% or more ClO₄<SUP>-</SUP> removal efficiency after a 7-day incubation when the initial concentration was approximately 120 mg ClO₄<SUP>-</SUP>/l. Genomic DNA was isolated from the enriched culture and its inoculum (activated sludge), and used for PCR-DGGE analysis of 16S rRNA genes. Microbial compositions of the enrichment culture and the activated sludge were different, as determined by their different DGGE profiles. The difference in DGGE banding patterns suggests that environmental conditions of the enrichment culture caused a change in the microbial community composition of the inoculated activated sludge. Dominant DGGE bands in the enrichment culture sample were affiliated with the classes β-Proteobacteria, Bacteroidetes, and Spirochaetes. Further investigation is warranted to reveal the metabolic roles of the dominant populations in the ClO₄<SUP>-</SUP> degradation process, along with their isolation.

PCR-DGGE and Real-time PCR dsrB-based Study of the Impact of Heavy Metals on the Diversity and Abundance of Sulfate-reducing Bacteria

Islamud-Din,Abd El-Latif Hesham,Ayaz Ahmad,Cang Daqiang,Sardar Khan 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.4

Sulfate-reducing bacteria (SRB) are widelyused for heavy metal (HM) treatment in bioreactors buttheir growth and biological activity can be inhibited bysuch treatment. Here, bioreactor experiments were used toinvestigate changes in the SRB community and the copynumber of the dissimilatory sulfite reductase β-subunitfunctional gene (dsrB) under high doses of sulfates andHMs. The SRB community was investigated using polymerasechain reaction denaturing gradient gel electrophoresis(PCR-DGGE) and sequencing techniques, while the dsrBgene abundance was measured by quantitative real-timePCR (qRT-PCR). The sulfate reduction rate was initiallymuch higher in reactors without HMs than in thosecontaining HMs (p = 0.001). Sulfate levels were reducedby 50% within the first 3 days of operation. As a result, theHM removal rate was initially much lower in the reactorscontaining HMs. Most of the HMs reduced to safe limitswithin 9 ~ 12 days of operation. The SRB communitymainly consisted of Desulfovibrio vulgaris, D. termitidis,D. desulfuricans, D. simplex and Desulfomicrobium baculatum,as determined by PCR-DGGE. qRT-PCR revealed adecreasing trend in the copy numbers of a functional gene(dsrB) after 6 days in samples lacking HMs; however, theopposite trend was observed in the HM-containing samples.

PCR-DGGE as a Supplemental Method Verifying Dominance of Culturable Microorganisms from Activated Sludge

( Sheng Zhou ),( Hai Wei Chao ),( Ke Lin ),( Hai Zhen Wu ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.11

To verify the dominance of microorganisms in wastewater biological treatment, PCR-DGGE (denaturing gradient gel electrophoresis) was performed as a supplementary support method for screening of the dominant microorganisms from activated sludge. Results suggest that the dominant microorganisms in activated sludge are primarily responsible for strengthening its effectiveness as a biological treatment system, followed by the non-main dominant microorganisms, whereas the non-dominant microorganisms showed no effects. The degree of microbial abundance present on the profile of PCR-DGGE was in line with the treatment efficiency of augmented activated sludge with isolated cultures, suggesting that PCR-DGGE can be used as an effective supplementary method for verifying culturable dominant microorganisms in activated sludge of coking wastewater.

식품내 미생물 동정을 위한 DGGE와 TGGE의 응용

박성희,김근성 중앙대학교 유전공학연구소 2003 遺傳工學硏究論集 Vol.16 No.1

분자생물학적 기법중 미생물군의 분석에 유용하게 이용되고 있는 DGGE(denaturing gradient gel electrophoresis)와 TGGE(tempertature gradient gel electrophoresis)의 기술에 대한 원리와 식품의 발효과정 중 변화되는 미생물 동정 사례를 고찰하였다. 이 기법들은 PCR 산물을 염기서열 분석 이전에 미생물군의 유전자 fingerprinting을 통하여 분석함으로서 배양에 의존하는 기존의 미생물학적 방법들 보다 빠르면서도 높은 감도를 통하여 효율적으로 분석할 수 있다. 그러므로 DGGE/TGGE 기술은 식품내에 존재하는 분리 될 수 있는 다양한 세균 균주의 동정에 이용될 수 있는 최근에 개발된 분자생물학적 기법이다. We describe the theoretical and practical aspects of DGGE(denaturing gradient gel electrophoresis) and TGGE(tempertature gradient gel electrophoresis) and their applications to the analysis of food microbial communities. DGGE or TGGE is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. Genetic fingerprinting techniques including DGGE or TGGE of PCR-amplified DNA fragments are more sensitive, rapid and effective than phenotypic techniques including traditional laboratory cultivation. Therefore, PCR-DGGE or TGGE techniques will be more frequently chosen as a new tool to differentiate diverse foodborne bacterial strains from each other.