16S rDNA 유전자를 이용한 Bordetella 근연종으로부터 Bordetella pertussis의 감별 PCR

정상운,문유미,성화영,강연호,유재연 대한감염학회 2008 Infection and Chemotherapy Vol.40 No.1

Background : Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. Materials and Methods : The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. Results : As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. Conclusion : In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed. 목 적 : 백일해의 원인균인 B. pertussis는 성장속도가 늦기 때문에 PCR 검출방법은 백일해 진단의 초기단계에서 유용하다. 현재 BP primer가 PCR 검출에 광범위하게 사용되나 BP primer는 최근에 B. holmesii와의 교차반응성이 보고되었다. 따라서 PCR 단계에서 B. pertussis와 B. holmesii의 감별이 필요하다. 이러한 이유로 본 논문에서는 16S rDNA sequence를 기반으로 하는 진단용 primer 를 새로 고안하였고, 그 효용성을 측정하였다. 재료 및 방법 : 특이적 PCR primer들은 다양한 Bordetella 속의 16S rDNA 유전자들의 정렬된 서열 집합체로부터 고안되었다. 고안된 primer의 특이도는 임상적으로 중요한 4개 Bordetella 종 (B. pertussis, B. holmesii, B. parapertussis, B. bronchiseptica)을 사용하여 평가하였다. 민감도 또한 평가하였고 그 결과를 BP primer와 비교하였다. 결 과 : 결과적으로, 새롭게 개발된 primer는 B. holmesii를 포함하는 다른 Bordetella 종들로부터 B. pertussis를 성공적으로 감별하였다. 민감도분석에서는 B. pertussis에 대한 16S-F2/16S-R1 primer의 검출한계는 5 pg의 genomic DNA와 105 cells/mL의 세포부유액까지 였다. 또한 임상검체에 대해서 BP primer set와 동일한 결과가 확인되었다. 결 론 : 본 연구에서 오직 B. pertussis에 대해 특이적인 PCR primer를 16S rDNA를 기반으로 하여 개발하였고, 이는 B. holmesii와의 교차반응성을 나타내지 않았다. 또한 본 연구에서 새롭게 개발된 primer의 임상검체에 대한 적용성도 확인되었다.

Vibrio 임상 분리주의 균종 동정을 위한 dnaJ 및 16S rDNA의 서열 분석법 비교

최인선,문대수,박건,강성호,김춘미,안영준,김동민,윤나라,임동훈,신성희,국중기,장영효,장숙진 대한진단검사의학회 2018 Laboratory Medicine Online Vol.8 No.1

배경: Vibrio 종에는 치명적인 패혈증을 일으키는 균종들도 포함되어 있어서 정확한 균종 동정이 매우 중요하다. 일부 비전형적인표현형을 보이는 균종이 있어 정확한 동정을 위해 적절한 분자 진단법의 도입이 필요하다. 방법: 본 연구에서는 Vibrio 임상 분리주 53주와 표준균주 8주가이용되었다. 분자 진단법으로는 dnaJ 유전자 서열 분석, 16S rDNA 서열 분석, gyrB V. vulnificus–특이적 PCR 서열 분석, gyrB V. navarrensis– 특이적 PCR 서열 분석, V. vulnificus hemolysin 유전자(vvh) PCR (V. vulnificus-특이적 PCR)를 시행하였다. 또한 16S rDNA 와 dnaJ 유전자, gyrB 유전자의 서열분석 결과를 토대로 계통수분석을 실시하였으며 상기 검사 결과를 종합하여 균의 최종 동정명이 결정되었다. 16S rDNA 서열 분석과 dnaJ 유전자 서열 분석 간의 일치율 분석은 카이 제곱 검정을 이용하였다. 결과: 61주의 Vibrio 균주의 최종 균종명 분포를 내림차순으로 배열하면 다음과 같다: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), Grimontia hollisae (1.64%). dnaJ 유전자 서열 분석의 정확도는 91.80%였고16S rDNA 서열 분석의 정확도는 86.89%였다. dnaJ 유전자 서열 분석법과 16S rDNA 서열 분석법 간의 일치도는 0.45로서, 중등도의일치도였다. 결론: 본 연구는 dnaJ 유전자 서열 분석법이 서로 밀접히 관련된Vibrio 종 간의 정확한 균 종정에 유용한 방법이라는 것을 보여주었다. Background: Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis. Methods: Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus–specific sequence, gyrB V. navarrensis–specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test. Results: The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement. Conclusions: Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene

Jung, Sang-Oun,Moon, Yu-Mi,Sung, Hwa Young,Kang Yeon Ho,Yu, Jae-Yon 대한감염학회 2008 감염과 화학요법 Vol.40 No.1

Background : Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. Materials and Methods : The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. Results : As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 10^5 cells/mL of cell suspension, In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. Conclusion : In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed. 목적 : 백일해의 원인균인 B. pertussis는 성장속도가 늦기 때문에 PCR 검출방법은 백일해 진단의 초기단계에서 유용하다. 현재 BP primer가 PCR 검출에 광범위하게 사용되나 BP primer는 최근에 B. holmesii와의 교차반응성이 보고되었다. 따라서 PCR 단계에서 B. pertussis와 B. holmesii이 감별이 필요하다. 이러한 이유로 본 논문에서는 16S rDNA sequence를 기반으로 하는 진단용 primer를 새로 고안하였고, 그 효용성을 측정하였다. 재료 및 방법 : 특이적 PCR primer들은 다양한 Bordetella 속의 16S rDNA 유전자들의 정렬된 서열 집합체로부터 고안되었다. 고안된 primer의 특이도는 임상적으로 중요한 4개 Bordetella 종 (B. pertussis, B. holmesii, B. parapertussis, B. bronchiseptica)을 사용하여 평가하였다. 민감도 또한 평가하였고 그 결과를 BP primer와 비교하였다. 결과 : 결과적으로, 새롭게 개발된 primer는 B. holmesii를 포함하는 다른 Bordetella 종들로부터 B. pertussis를 성공적으로 감별하였다. 민감도분석에서는 B. pertussis에 대한 16S-F2/16S-R1 primer의 검출한계는 5 pg의 genomic DNA와 10^5 cells/mL의 세포부유액까지 였다. 또한 임상검체에 대해서 BP primer set와 동일한 결과가 확인되었다. 결론 : 본 연구에서 오직 B. pertussis에 대해 특이적인 PCR primer를 16S rDNA를 기반으로 하여 개발하였고, 이는 B. holmesii와의 교차반응성을 나타내지 않았다. 또한 본 연구에서 새롭게 개발된 primer의 임상검체에 대한 적용성도 확인되었다.

Molecular Systematics of the Tephritoidea (Insecta: Diptera): Phylogenetic Signal in 16S and 28S rDNAs for Inferring Relationships Among Families

Han, Ho-Yeon,Ro, Kyung-Eui,Choi, Deuk-Soo,Kim, Sam-Kyu The Korean Society for Integrative Biology 2002 Korean journal of biological sciences Vol.6 No.2

Phylogenetic signal present in the mitochondrial 16S ribosomal RNA gene (16S rDNA) and the nuclear large subunit ribosomal RNA gene (28S rDNA) was explored to assess their utility in resolving family level relationships of the superfamily Tephritoidea. These two genes were chosen because they appear to evolve at different rates, and might contribute to resolve both shallow and deeper phylogenetic branches within a highly diversified group. For the 16S rDNA data set, the number of aligned sites was 1,258 bp, but 1,204 bp were used for analysis after excluding sites of ambiguous alignment. Among these 1,204 sites, 662 sites were variable and 450 sites were informative for parsimony analysis. For the 28S rDNA data set, the number of aligned sites was 1,102 bp, but 1,000 bp were used for analysis after excluding sites of ambiguous alignment. Among these 1000 sites, 235 sites were variable and 95 sites were informative for parsimony analysis. Our analyses suggest that: (1) while 16S rDNA is useful for resolving more recent phylogenetic divergences, 28S rDNA can be used to define much deeper phylogenetic branches; (2) the combined analysis of the 16S and 28S rDNAs enhances the overall resolution without losing phylogenetic signal from either single gene analysis; and (3) additional genes that evolve at intermediate rates between the 16S and 28S rDNAs are needed to further resolve relationships among the tephritoid families.

Molecular Identification of Anginosus Group Streptococci Isolated from Korean Oral Cavities

Soon-Nang Park,Mi-Hwa Choi,Joong-Ki Kook The Korean Academy of Oral Biology 2013 International Journal of Oral Biology Vol.38 No.1

Anginosus group streptococci (AGS) were classified based on the nucleotide sequences of the 16S rRNA gene (16S rDNA) and comprised Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus. It is known that AGS is a causative factor of oral and systematic diseases. The purpose of this study was to discriminate the 56 clinical strains of AGS isolated from Korean oral cavities using phylogenetic analysis of 16S rDNA and species‐specific PCR at the species-level. The 16S rDNA of clinical strains of AGS was sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. PCR was performed to identify the clinical strains using species‐specific primers described in previous studies and S. intermedius‐specific PCR primers developed in our laboratory. The resulting phylogenetic data showed that the 16S rDNA sequences can delineate the S. anginosus, S. intermedius, and S. constellatus strains even though the 16S rDNA sequence similarity between S. intermedius and S. constellatus is above 98%. The PCR data showed that each speciesspecific PCR primer pair could discriminate between clinical strains at the species‐level through phylogenetic analysis of 16S rDNA nucleotide sequences. These results suggest that phylogenetic analysis of 16S rDNA and PCR are useful tools for discriminating between AGS strains at the species-level.

16S rDNA-RFLP에 의한 Spirastrella abata와 Spirastrella panis 해면에 서식하는 배양가능한 공생세균 군집의 비교

조현희,박진숙,Cho, Hyun-Hee,Park, Jin-Sook 한국미생물학회 2009 미생물학회지 Vol.45 No.2

계통적으로 근연하며 지리적 분포가 유사한 두 종의 Spirastrella 속의 해양 해면, S. panis와 S. abata의 배양 가능한 공생세균 군집구조를 16S rDNA-RFLP 방법에 의해 분석하였다. 공생세균의 배양은 해면추출물 3%를 포함하는 MA 배지를 사용하였다. 증폭된 16S rDNA의 RFLP (restriction fragment length polymorphism) 분석을 위한 제한효소로 HaeIII와 MspI을 이용하였으며, 그 결과 24개의 RFLP type을 구별할 수 있었다. 각 패턴별로 1~5개의 분리균주를 선별하여 부분 염기서열 분석 결과, 알려진 세균 종과 98.4% 이상의 유사도를 나타내었으며 2종의 Spirastrella 해면으로부터 분리된 세균들은 모두 Alphaproteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteria 4개의 강(class)에 포함되었다. Alphaproteobacteria는 S. abata에서 39.3%, S. panis에서 47.6%가 관찰되어 두 해면에서 우점하는 세균 군집이었다. Gammaproteobacteria의 경우 S. abata에서 38.5%로 관찰된 반면 S. panis에서 1.6%의 아주 적은 비율로 관찰되었다. 또한 Bacillus (phylum Firmicutes) 종은 S. abata에서 9.7%를 나타낸 반면, S. panis에서는 44.3%의 분포를 나타내었다. Planococcus maritimus (8.1%, phylum Firmicutes)와 Psychrobacter nivimaris (28.9%, phylum Gammaproteobacteria)는 S. abata에서만 관찰되어 이들은 S. abata에 특이적인 세균 종임을 알 수 있었다. 같은 장소에 서식하는 계통적으로 근연한 두 종의 해면에서 공생세균의 군집 구조는 차이가 큰 것으로 나타났다. A cultivation-based approach was employed to compare the culturable bacterial diversity associated with two phylogenetically closely related marine sponges, Spirastrella abata and Spirastrella panis, which have geologically overlapping distribution patterns. The bacteria associated with sponge were cultivated using MA medium supplemented with 3% sponge extracts. Community structures of the culturable bacteria of the two sponge species were analyzed with PCR-RFLP (restriction fragment length polymorphism) based on 16S rDNA sequences. The RFLP fingerprinting of 16S rDNA digested with HaeIII and MspI, revealed 24 independent RFLP types, in which 1-5 representative strains from each type were partially sequenced. The sequence analysis showed >98.4% similarity to known bacterial species in public databases. Overall, the microbial populations of two sponges investigated were found to be the members of the classes; Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria. The Alphaproteobacteria were predominant in the bacterial communities of the two sponges. Gammaproteobacteria represented 38.5% of bacterial community in S. abata. Whereas only 1.6% of this class was present in S. panis. Bacillus species were dominat in S. panis. Bacillus species were found to be 44.3% of bacterial species in S. panis, while they were only 9.7% in S. abata. It is interesting to note that Planococcus maritimus (8.1%, phylum Firmicutes) and Psychrobacter nivimaris (28.9%, phylum Gammaproteobacteria) were found only in S. abata. This result revealed that profiles of bacterial communities from the sponges with a close phylogenetic relationship were highly species-specific.

16S rDNA-ARDRA법을 이용한 소나무림과 상수리나무림 토양 내 VBNC 세균군집의 계통학적 특성 비교

한송이,김윤지,황경숙,Han Song-Ih,Kim Youn-Ji,Whang Kyung-Sook 한국미생물학회 2006 미생물학회지 Vol.42 No.2

직접 생균수 측정법(DVC)과 평판계수법(PC)을 이용하여 소나무림과 상수리나무림 토양에 분포하는 세균군집의 정량적 평가를 실시한 결과, DVC법에 의해 계수된 생균수에 대해 평판법에 의해 계수된 생균수 1% 미만으로 나타났다. 이상의 결과로부터 산림토양 내에 평판배양법으로는 배양이 곤란한 난배양성(viable but non culturable; VBNC) 세균이 99% 이상 존재해 있는 것으로 판단되었다. 이들 VBNC 세균의 군집구조 해석을 위하여 토양으로부터 직접 DNA를 추출하고 16S rDNA-ARDRA 분석을 통하여 계통학적 특성을 검토하였다. 소나무림과 삼수리나무림 토양으로부터 각각 111 clones, 108 clones을 획득하고 HaeIII 절편양상에 따라 30 groups과 26 groups의 ARDRA group으로 분류하였다. 각 ARDRA group으로부터 대표 clone을 선발하여 16S rDNA 염기서 열을 결정한 결과, 소나무림 토양의 경우 ${\alpha}$-proteobacteria (12 clones), ${\gamma}$-proteobacteria (3 clones), ${\delta}$-proteobncteria (1clone), Flexibacter/Cytophaga (1 clone), Actinobacteria (4 clones), Acidobacteria (4 clones), 그리고 Planctomycetes (5 clone)의 7개의 계통군이 확인되었으며, 상수리나무림 토양에서는 ${\alpha}$-proteobacteria (4 clones), ${\gamma}$-proteobacteria (2 clones), Actinobacteria (10 clones), Acidobacteria (8 clones), Planctomycetes (1 clone), 그리고 Verrucomicrobia (1clone)로 6개의 다양한 계통군이 확인되었다. 이상, 소나무림과 상수리나무림 토양 내에 존재하는 99% 이상의 VBNC 세균군집의 대부분은 미배양성 혹은 미동정균으로 계통학적으로 다양한 미지의 미생물로 구성되어 있음이 확인되었다. In this study was performed to analyze quantitatively the number of viable but non-culturable bacteria in the Pine and Quercus forest soil by improved direct viable count (DVC) and plate count (PC) methods. The number of living bacteria of Pine and Quercus forest soil by PC method were less then 1% of DVC method. This result showed that viable but non-culturable (VBNC) bacteria existed in the forest soil with high percentage. Diversity and structure of VBNC bacterial populations in forest soil were analyzed by direct extracting of DNA and 16S rDNA-ARDRA from Pine and Quercus forest soil. Each of them obtained 111 clones and 108 clones from Pine and Quercus forest soil. Thirty different RFLP types were detected from Pine forest soil and twenty-six different RFLP types were detected from Quercus forest soil by HeaIII. From ARDRA groups, dominant clones were selected for determining their phylogenetic characteristics based on 16S rDNA sequence. Based on the 16S rDNA sequences, dominant clones from ARDRA groups of Pine forest soil were classified into 7 major phylogenetic groups ${\alpha}$-proteobacteria (12 clones), ${\gamma}$-proteobacteria (3 clones), ${\delta}$-proteobacteria (1 clone), Flexibacter/Cytophaga (1 clone), Actinobacteria (4 clones), Acidobacteria (4 clones), Planctomycetes (5 clones). Also, dominant clones from ARDRA groups of Quercus forest soil were classified into 6 major phylogenetic groups : ${\alpha}$-proteobacte,ia (4clones), ${\gamma}$-proteobacteria (2 clones), Actinobacteria (10 clones), Acidobacteria (8 clones), Planctomycetes (1 clone), and Verrucomicobia (1 clone). Result of phylogeneric analysis of microbial community from Pine and Quercus forest soils were mostly confirmed at uncultured or unidentified bacteria, VBNC bacteria of over 99% existent in forest soil were confirmed variable composition of unknown micro-organism.

화학무기영양성 질화세균의 생리학적 탐색 및 분자적 동정

정필문,박성주 대전대학교 기초과학연구소 1999 自然科學 Vol.10 No.1

폐수처리장의 확성슬러지 시료에서 화학무기영양성 질화 세균을 무기염류선택배지에서의 배양을 통한 생리학적 방법으로 분리하여 순수배앙한 다음 이를 분자생물학적 방법으로 동정하였다. 선택배지에서 자란 50개의 콜로니를 다시 영양배지에서 배양하여 콜로니를 형성하지 못한 13개의 콜로니를 선별하였다. 이 가운데 계대배양에 성공한 4개의 균주를 농화배앙하여 16 rDNA 염기서열 분석을 통하여 질화 세균을 동정하였다. 16S rDNA의 증폭을 위한 CNA는 세균의 CNA를 추출하는 대신 순수배양된 콜로니 세포를 사용하였으며 여기에는 진정세균의 universal primer가 사용되었다. 증폭된 16S rDNA 를 RFLP(restriction fragment length polymorphism)에 의하여 상동성을 분석한 결과 4종류 서로 다른 미생물로 판단되었다. 4종류의 16S rDNA 염기서열은 National Center for Biotchnology lnformation(NCBI) 의 BLAST2.0 프로그램을 이용하여 동정한 결과 1종류는 Nitrobacter 16S rDNA와 96%의 일치도를 보였으며 나머지는 데이터베이스에는 없는 염기서열로 확인되었다. A culture-dependent molecular identification approach was used to isolate and enrich chemolithotrophic nitrifying bacteria from activated sludge of a wastewater, treatment plant. Fifty bacterial colonies were screened on selective media, and selected 13 of which were screened by inability of growth in nutrient media. Nine of thirteen screened isolates failed to grow during the sub-culture procedure, and only four pure-cultured isolates were identified y partial 16S rDNA sequencing. 16S rDNA of enriched isolates were amplified directly from colonies without DNA extraction usin universal Bacteria-specific primers. The sequence homology of four PCR products was compared by restriction fragment length polymorphism (RFLP), and all of which were unique. These four types of sequences were determined and identified using BLAST 2.0 program of National Center for Biotechnology Information. One of the 4 sequences identified was 96% identical to Nitrobacter 16S rDNA sequences and the others were not matched to any known nitrifying bacterial 16S rDNA sequences.

혈액 배양 액체배지에서 세균성 병원균 검출을 위한 16S rDNA PCR의평가

장숙진,김진희,김영숙,신종희,박건,비두루,문대수,박영진 대한임상미생물학회 2006 Annals of clinical microbiology Vol.9 No.1

Background: Rapid detection of pathogens in blood is important in patient management, because the mortality rate associated with bloodstream infections is very high. We evaluated the efficiency of a 16S rDNA PCR assay for the detection of various pathogens in blood culture broth in a clinical laboratory. Methods: 16S rDNA PCR was performed on 221 blood culture bottles consisting of 99 culturepositive and 122 culture-negative samples. The results were compared with conventional culture methods. We also compared the efficiency of three DNA extraction and purification methods using proteinase K, triton X-100, and benzyl alcohol-guanidine DNA extraction of blood culture broths. Results: The 16S rDNA PCR method detected 95 (12 Staphylococcus aureus, 27 coagulase negative staphylococci, 10 enterococci, 5 streptococci, 37 gram negative bacilli, 4 corynebacteria) of 99 positive culture bottles. Four false-negative results were obtained for bottles containing 2 Corynebacterium, 1 Escherichia coli, and 1 S. aureus species. All 122 bottles that showed no blood culture growth were negative by 16S rDNA PCR. Overall, the sensitivity, specificity, positive predictive values and negative predictive values of 16S rDNA PCR relative to the culture results were 96.0%, 100%, 100%, and 96.8%, respectively. Among the three DNA extraction methods, the benzyl alcohol-guanidine method was most effective. Conclusion: The 16S rDNA PCR assay is a rapid and efficient means of detecting various pathogens in the blood and has great potential for use in the clinical microbiology laboratory.

16S rRNA 유전자 분석에 의한 전남 순천만 갯벌의 세균 다양성

이명숙,홍순규,이동훈,배경숙 한국미생물학회 2001 미생물학회지 Vol.37 No.2

순천만 갯벌의 세균 군집의 다양성을 조사하기 위해 16S rDNA의 다양성을 조사하였다. 갯벌로부터 전체 핵산을 분리한 후, 세균에 상보적인 universal primer로 증폭된 16S rDNA로부터 클론 라이브러리를 만들었다. 총 111개의 클론으로부터 HaeIII를 이용하여 amplified rDNA restriction analysis (ARDRA)를 수행하고, Gelcompar II 프로그램을 이용하여 pattern을 clustering하였다. 111개의 클론 중 100가지의 서로 다른 RFLP type이 조사되었고, 이들 중 전체 클론 라이브러리를 대표할 수 있는 20개의 클론을 선별하여 부분적인 염기서열을 분석하여 세균 다양성을 분석하였다. 20개의 클론중에는 RDP와 GenBank에서 제공하는 small subunit RNA database와 동일한 클론은 존재하지 않았으며, 이미 알려진 배양 가능한 세균의 16S rRNA 염기서열과 비교 하였을때 77∼96.8%의 유사도를 보였다. 또한 이들 20개의 클론은 alpha-, delta-, gamma-Proteobacteria, low G+C Gram positive bacteria, high G+C Gram positive bacteria, Sphingobacteria (Cytophaga); Cyanobacteria (Chloroplast)등 주요한 7개 lineage에 속했으며, 클론들 중 Proteobacteria가 우점종을 차지하고 있었다. In order to investigate the diversity of bacterial community in the mud flat of Sunchon Bay, Chunnam province, diversity of amplified 16S rDNA was examined. Total DNA was extracted from sediment soils and 16S rDNAs were amplified using PCR primers based on the universally conserved sequences in bacteria. Clonal libraries were constructed and 111 clones were examined by amplified rDNA restriction analysis (ARDRA) using HaeIII. Clones were clustered based on restriction patterns using computer program, GelCompar II. One hundred different RFLP types were detected from 111 clones. The 20 clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the bacterial diversity in the clone libraries. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA databases and GenBank. All sequences showed between 77 and 96.8% similarity to the known 16s rRNA sequence from cultured organisms. The 20 clones sequenced fell into seven major lineages of the domain Bacteria: alpha-, delta-, gamma-Proteobacteria, low G+C Gram positive bacteria, high G+C Gram positive bacteria, Sphingobacteria (Cytophaga) and Cyanobacteria (chloroplast). Among the clones, the Proteobacteria were dominant.