• 한국미생물·생명공학회지
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• 제36권4호
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• pp.247-259
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• 2008

미생물 구조 및 집단 구성원의 변화를 배양에 의존하지 않고, 빠르게 평가할 수 있는 방법의 하나인 terminal-restriction fragment length polymorphism (I-RFLP) 분석을 김치 유산균 연구에 적용하여, $15^{\circ}C$ 및 $4^{\circ}C$ 발효 김치에 관여하는 유산균의 다양성과 역동성을 검토하였다. 두 발효온도에서 공통적으로 Leuconostoc mesenteroides, Lc, inhae, Lc. kimchii, Weissella koreensis, W. cibaria, Lactobacillus sakei, Lb. curvatus, Lb. plantarum, Lb. paraplantarum, Lb. pentosus, 및 Lb. brevis의 존재가 예상되었고, Lc. citreum과 Enterococcus faecalis는 각각 $15^{\circ}C$와 $4^{\circ}C$에서 검출되었다. W. koreensis는 중기발효에 우점을 점하였고, Lactobacillus 속은 발효 후기의 우점종으로 나타났다. Lb. sakei, Lb. curvatus는 발효온도에 관계 없이 우점종을 형성하였지만, Lb. plantarum, Lb. paraplantarum, Lb, pentosus와 Lb. brevis의 생육은 저온에서 잘 일어나지 않았다. 몇 종의 Leuconostoc 속 유산균은 발효 후기까지 생장이 유지되었다.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.15-21
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• 2002

Terminal-restriction fragment length polymorphism (T-RFLP) analysis has been optimized by using in vitro model community composed of genomic DNAs of known bacterial strains and has been applied to assess the bacterial community structure in marine sediments. The specific fluorescence-labeled terminal restriction fragments (T-RFs) between 39 and 839 base long specifying each strain were precisely measured for known bacterial strains. The addition of a co-solvent (dimethylsulfoxide or glycerol) into PCR reactions has reduced differential PCR amplification. Comparative bacterial community structure was investigated for pristine and polluted sediments. A complex T-RFLP pattern showing complex bacterial community structure was obtained in the pristine sediment, whereas simple T-RFLP pattern (low bacterial diversity) was shown in polluted sediments where caged aquaculture has been conducted for several years. The results of T-RFLP analysis were compared with that of cloning and sequencing 16S rDNA clones from the same sediments. Sequence analysis of 16S rDNA clones (72) of the pristine sediment revealed a diverse collection of lineages, largely of the class Proteobacteria ($6\%$ alpha subdivision, $46\%$ gamma subdivision, $13\%$ delta subdivision, and $3\%$ epsilon subdivision), Nitrospina $(8\%)$, high G+C gram positive $(8\%)$, Verrucomicrobia $(7\%)$, and Planctomycetes $(6\%)$. In the contaminated sediments, 17 $(59\%)$ of the 16S rDNA clones (29) were related to Campylobacter and symbiont of Rimicaris exoculata belonging to epsilon subdivision of Proteobacteria. The results obtained indicated that T-RFLP analysis is a rapid and precise technique for comparative bacterial community analysis.

• 한국식물병리학회지
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• 제12권2호
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• pp.162-168
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• 1996

우리 나라의 주요 고추 재배지와 미국, 대만, 호주, 아르헨티나에서 수집된 44 개 고추 더뎅이병균(Xanthomonas campestris pv. vesicatoria)균주간의 유전적변이를 genomic DNA의 restriction fragment length polymorphism(RFLP)에 의해 분석하였다. Genomic DNA RFLP profiles을 cluster 분석하여 얻은 dendrogram에서 지리적 기원이 다른 44개 균주들은 11개 RFLP 그룹으로 분류되었다. 외국 균주들은 genomic DNA의 RFLP 분석에 의해 모두 각각 다른 RFLP 그룹으로 분류되었다. 외국 균주들 중에서 미국 균주는 우리 나라 일부 균주들과 밀접한 유전적 관련성을 가지고 함께 cluster를 이루었는데, 이것은 이 균주들이 동일한 고추 더뎅이병균의 조상 균주 집단에서 유래했으리라는 것을 시사해 준다. 우리 나라 균주들은 6개의 RFLP 그룹으로 분류되었다. 대부분의 우리 나라 균주들은 가까운 cluster를 이루며 미국 균주를 제외한 외국 균주들과 뚜렷하게 구분되었다. 그러나 우리 나라 균주들 중에서 마산에서 수집된 Ms93-1은 다른 우리 나라 균주들과 뚜렷하게 구분되었다. 유전적으로 격리된 균주의 출현은 우리 나라에서 지리적 기원이 다른 고추 더뎅이병균 균주 사이에 이미 발생한 다양한 유전적 분화의 결과라고 추론된다.

• 한국미생물·생명공학회지
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• 제40권4호
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• pp.389-395
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• 2012

배양 비의존적 미생물 군집분석법의 하나로 활발하게 이용되고 있는 T-RFLP (terminal restriction fragment length polymorphism) 분석과 실험동물을 이용한 생균제의 장관 정착 평가를 시도해 보았다. 장관 정착 평가를 위한 생균제는 cinnamoyl esterase 활성 보유 유산균을 발효 침채류로부터 분리하였고, 16S ribosomal RNA 유전자 염기서열 분석을 통해 동정하였다. 분리 균주 중, cinnamoyl esterase에 의한 chlorogenic acid 분해활성이 높고, 내산성 및 담즙산 내성의 평가 결과가 우수한 Lactobacillus plantarum KK3 균주를 생균제로 선정하였다. 배양 후, 냉동건조하여 제조한 생균제를 생쥐에게 투여한 다음, T-RFLP 분석을 이용하여 생쥐 분변의 미생물상을 모니터링하였다. 생균제 투여에 따라 검출되기 시작한 L. plantarum의 T-RF가 투여 중지 후 24일까지 지속됨을 확인하여 T-RFLP 분석법이 생균제로 사용한 L. plantarum의 장관 정착 평가에 유용한 것으로 나타났다. 생균제 투여 전 생쥐의 분변에서 검출되지 않았던 cinnamoyl esterase 활성 보유 L. plantarum이 투여 중지 후 24일차 분변에서 배양법으로 검출되어 L. plantarum KK3 균주의 생쥐 장관 정착이 확인되었다.

• 대한임상검사과학회지
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• 제38권3호
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• pp.158-165
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• 2006

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

• 환경생물
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• 제36권3호
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• pp.352-358
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• 2018

본 연구는 PCR 기반 RFLP (Restriction Fragment Length Polymorphism; 제한절편 길이 다형성) 분자기법을 활용하여 난 및 치어 대상 납자루아과 어류 3종의 동정을 좀 더 빠르고 정확하게 파악하고 납자루아과 어류의 종별 산란양상 및 번식생태 이해에 대한 기여가 목적이다. 본 연구를 위해 기존 선행된 문헌자료를 확인하고 납자루아과 어류가 2종 이상 동서하고 있는 지역을 확인하여 현지조사를 수행하였다. 현지조사 결과 확인된 납자루아과 어류는 묵납자루(Acheilognathus signifer), 줄납자루(A. yamatsutae) 및 각시붕어(Rhodeus uyekii)로 총 3종이 확인되었으며, 확인된 납자루아과 어류와 동서하고 있는 숙주조개(작은말조개; Unio douglasiae sinuolatus)를 채집하여 숙주조개 속 납자루아과 어류의 난 및 치어를 확보하였다. 현지조사 결과 확인된 납자루아과 어류 3종을 대상으로 미토콘드리아 DNA COI과 cyt b 유전자 염기서열을 비교하여 각각 종별로 특이성을 지닌 부위(단일염기변이; Single Nucleotide Variation: SNV)에 맞는 제한효소를 선정하였고, 숙주조개 속 난 및 치어를 대상으로 genomic DNA를 추출하여 PCR-RFLP 실험을 수행한 결과 현지조사 시 확인된 납자루아과 어류 3종의 독특한 제한절편 길이 양상을 전기영동을 통하여 확인하였다. 본 연구를 통해 묵납자루, 줄납자루 및 각시붕어의 종을 판별할 수 있는 RFLP 마커를 개발하였으며, 숙주조개 난 및 치어를 대상으로 정확한 종의 동정을 보다 빠르고 효과적으로 수행하여 각각 납자루아과 종별 산란양상을 보다 정확히 규명하고 향후 이들 자연개체군의 효과적인 유지, 관리 및 보전 방법 개발에 유용하게 활용될 수 있을 것으로 판단된다.

• Journal of Microbiology
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• 제38권2호
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• pp.66-73
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• 2000

To investigate the genetic relationship among 12 species belonging to the Fusarium section Martiella, Dlaminia, Gibbosum, Arthrosporiella, Liseola and Elegans, the internal transcribed spacer(ITS) regions of ribosomal DNA (rDNA) were amplified with primer pITS1 and pITS4 using the polymerase chain reaction(PCR). After the amplified products were digested with 7 restriction enzymes, restriction fragment length polymorphism (RFLP) patterns were analyzed. The partial nucleotide sequences of the ITS region were determined and compared. Little variation was observed in the size of the amplified product having sizes of 550bp or 570bp. Based on the RFLP analysis, the 12 species studied were divided into 5 RFLP types. In particular, strains belonging to the section Martiella were separated into three RFLP types. Interestingly, the RFLP type of F. solani f. sp. piperis was identical with that of isolates belonging to the section Elegans. In the dendrogram derived from RFLP analysis of the ITS region, the Fusarium spp. examined were divided into two major groups. In general, section Martiella excluding F. solani f. sp. piperis showed relatively low similarity with the other section. The dendrogram based on the sequencing analysis of the ITS2 region also gave the same results as that of the RFLP analysis. As expected, 5.8S, a coding region, was highly conserved, whereas the ITS2 region was more variable and informative. The difference in the ITS2 region between the length of F. solani and its formae speciales excluding F. solani f. sp. piperis and that of other species was caused by the insertion/deletion of nucleotides in positions 143-148 and 179-192.

• Animal cells and systems
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• 제7권2호
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• pp.145-149
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• 2003

Adult periodontitis is a chronic inflammatory disease whose etiology is not well defined. Recent studies have shown that vitamin D receptor gene has been a candidate for the susceptibility of adult periodontitis. The purpose of this study is to investigate the frequency of Taq I restriction fragment length polymorphism (RFLP) in the vitamin D receptor gene in nan periodontically healthy controls and 28 adult periodontitis patients. Taq I RFLP in the vitamin D receptor gene was detected by PCR amplification, followed by restriction enzyme digestion and 2％ agarose gel electrophoresis. There was no significant difference in the distribution of Taq I RFLP between healthy controls and adult periodontitis group (P > 0.05). Thus, Taq I RFLP in the vitamin D receptor gene may not confer the susceptibility to adult periodontitis in Korean population. However, t allele distributions of this RFLP showed various frequencies among ethnic groups studied. Further studies in other ethnic groups will be required.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.577-609
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• 1995

Bacteria have been regarded as one of the most important factors in pulpal and periapical diseases. Streptococci are frequently isolated facultative anaerobes in infected root canals. Recently molecular biological techniques have been rapidly progressed. This study was designed to apply the molecular biological tools to the identification and classification of streptococci in the endodontic microbiology. Streptococci isolated from infected root canals were identified with both Vitek Systems and API 20 STREP. Identification results were somewhat different in several strains of streptococci. Eighteen streptococci and enterococcal was difficult so to digest plasmid DNA using Hind III and EcoRI to differentiate strains by restriction enzyme analysis of plasmid DNA. 16S rDNA of chromosome was amplified by polymerase chain reaction(PCR) and then restricition fragment length polymorphism(RFLP) using several restriction enzymes was observed. The molecular mass of 16S rDNA of chromosomal DNA was approximately 1.4kb. There were three to five RFLP patterns using eight restriction enzymes. RFLP patterns digested with CfoI which recognizes four base sequences were identical in all stains. Hind III which recognizes six base sequences could not digest the 16S rDNA. Restriction enzymes which recognize five base sequences were suitable for RFLP pattern analysis. At least three different restriction enzymes were needed to compare each strains. 16S rDNA PCR-RFLP was simple and rapid to differentiate and classify strains and could be used in the epidemiological study of root canal infections.

• Asian-Australasian Journal of Animal Sciences
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• 제30권2호
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• pp.262-266
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• 2017

Objective: This study was conducted to screen scrotal hernia in domesticated swine from selected breeders in the Philippines. This defect is associated with a cytosine to thymine mutation in the BCL-2 associated X protein (BAX) gene of swine. Methods: Genetic screening was done by DNA extraction followed by amplification and digestion using polymerase chain reaction-restriction fragment length polymorphism, amplifying the 416 bp region of the BAX gene that was subjected to digestion using the Ear I enzyme. Sequencing was also conducted to validate the results. Results: Results revealed that out of 538 samples tested, 411 (76.4%) of the samples were found to be normal whereas the remaining were carriers of the mutation in which 80 (14.9%) were heterozygous mutants and 47 (8.7%) were homozygous mutants. Pietrain breed was found to have the highest incidence. Conclusion: Having a scrotal hernia eliminates the chances of using the boar as a breeder stock because the following generations arising from it would most likely exhibit herniation. It is therefore advised to establish a genetic screening method for Scrotal Hernia in the Philippines to eliminate the negative gene from the herd.