• International Journal of Oral Biology
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• 제35권1호
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• pp.21-25
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• 2010

This study was undertaken to develop species-specific forward and universal reverse PCR primers for the detection of Streptococcus sobrinus. These primers target the variable regions of the 16S ribosomal RNA coding gene (rDNA) and their specificity was tested against 10 strains of S. sobrinus strains and 20 different species of oral bacteria using serial dilutions of the purified genomic DNA of S. sobrinus ATCC $33478^T$. Our data show that species-specific amplicons were obtained from all the S. sobrinus strains tested but not from other species. Both direct and nested PCR could detect as little as 400 pg and 4 fg of genomic DNA from S. sobrinus ATCC $33478^T$, respectively. This result suggests that these PCR primers are highly specific and sensitive and applicable to the detection of S. sobrinus.

• 한국미생물·생명공학회지
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• 제39권1호
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• pp.70-76
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• 2011

Weissella 속 유산균 검출의 차이를 이용한 한국산 및 중국산 김치 판별의 가능성 검토를 위하여 Weissella 속 9종 균주의 PCR 검출법을 개발하였다. 종(species) 수준에서의 Weissella 속 균주의 특이적 PCR 검출을 위한 primer는 recN 유전자의 염기서열을 이용하여 선정하였으며, 김치로부터 W. cibaria, W. confusa, W. koreensis, W. soli를 모두 검출하기 위해서는 20 ng template DNA가 필요한 것으로 나타났다. 한국산 김치시료로부터는 W. cibaria, W. confusa, W. koreensis가 높은 빈도로 검출되었지만, W. soli는 검출되지 않았다. 한편 중국산 김치시료로부터는 이들 4종의 Weissella 속 균주들이 모두 검출되었다. 본 연구자들이 개발한 W. soli 특이적 PCR 검출은 현시점에서 중국산 김치의 원산지 판별법으로 적용되기에는 한계점을 가지고 있지만, 미생물 군집의 차이를 이용한 새로운 과학적 검증법이 제시되어 그 가능성이 검토되었다는 점에서 의의를 가지고 있다.

• The Plant Pathology Journal
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• 제31권3호
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• pp.212-218
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• 2015

In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP), X. hyacinthi (XH) and X. campestris pv. zantedeschiae (XCZ), based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of $1pg/{\mu}l$ per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

• Mycobiology
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• 제33권2호
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• pp.104-108
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• 2005

Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species.

• Fisheries and Aquatic Sciences
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• 제15권2호
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• pp.157-162
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• 2012

To analyze nucleotide sequence encoding internal transcribed spacer (ITS) regions specific to the Laminariaceae family, genomic DNA was isolated from six brown algae species distributed along the east coast of Korea. These included three species from the Laminariaceae family (Agarum clathratum Dumortier, Costaria costata [C. Agardh] Saunders, and Saccharina japonica Areschoug) and two species from the Alariaceae family (Undaria pinnatifida [Harvey] Suringer and Ecklonia cava Kjellman), both in the order Laminariales, and one species from the family Sargassaceae in the order Fucales (Sargassum serratifolium). Based on a sequence analysis of ITS-1 and ITS-2 for A. clathratum, C. costata, and E. cava, oligonucleotides were designed from the regions that showed sequence conservation in Laminariaceae. Following polymerase chain reaction using three sets of primers, amplification of ITS-1 and ITS-2 was detected in reactions using genomic DNA isolated from the species belonging to Laminariaceae, but not from the species belonging to the other families. The results indicate that this method can be used for the detection and identification of Laminariaceae species.

• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.273-277
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• 2013

Vibriosis caused by Vibrio anguillarum and edwardsiellosis caused by Edwardsiella tarda are septicemic diseases of many commercially important freshwater and marine fishes, and threaten the aquaculture industry in Korea. Early diagnosis and accurate identification of these two bacterial species could help to prevent these diseases and minimize the damage to cultured marine species. This study designed a duplex polymerase chain reaction (PCR) method for the simultaneous detection of two major fish pathogens: V. anguillarum and E. tarda. Each pair of oligonucleotide primers exclusively amplified the target groEL gene of the specific microorganism. Twenty-two Vibrio and ten non-Vibrio enteric species were used to check the specificity of the primers, which were found to be highly specific for the target species, even among closely related species. The detection limit was 400 pg for V. anguillarum and 4 ng for E. tarda when mixed purified DNA was used as the template. This assay showed high specificity and sensitivity in the simultaneous detection of V. anguillarum and E. tarda from artificially inoculated seawater and fish.

• Journal of Microbiology and Biotechnology
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• 제16권5호
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• pp.740-747
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• 2006

Amplification by universal consensus sequences in pathogenic bacterial DNA would allow rapid identification of pathogenic bacteria, and amplification of genus-specific and species-specific sequences of pathogenic bacterial DNA might be used for genotyping at the genus and species levels. For design of probes for molecular diagnostics, several tools are available as stand-alone programs or as Web application. However, since most programs can design only a few probe sets at one time, they are not suitable for large-scale and automatic probes design. Therefore, for high-throughput design of specific probes in diagnostic array development, an automated design tool is necessary. Thus, we developed a Web-based automatic system for design of genus-specific and species-specific probes for pathogen detection. The system is available at http://www.miprobe.com.

• 한국균학회지
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• 제44권2호
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• pp.103-107
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• 2016

본 연구는 국내 주요 식물검역관리 대상 Phytophthora pinifolia를 대상으로 신속하고 정확한 병원균 종동정 및 검출을 위해 Ypt1 유전자 염기서열을 활용하여 제작된 종 특이적 분석용 분자마커와 다양한 PCR 기법을 활용하여 병원균들에 대한 다양한 검출기술의 표준화 및 최적 검출 시스템 구축을 통하여 실제 검역검역 현장에서 활용 가능한 병원균 존재여부의 신속, 정확한 판별기법을 개발하고자 수행하였다. Ypt1 영역의 염기서열을 기초로 선발된 종 특이적 primer는 1~10 pg의 검출민감도를 가지며 193 bp의 종 특이적 PCR 증폭산물을 형성시켰다. 또한 선발된 종 특이적 primer의 종 특이성을 확인하기 위하여 국내 식물검역대상에 포함된 Phytophthora속 종들과 주요 식물병원균들을 대상으로 conventional PCR과 real-time PCR을 수행한 결과 목표로 한 P. pinifolia DNA에서만 특이적 PCR 증폭산물을 확인할 수 있었다. 선발된 종 특이적 primer에 대한 PCR의 검출 민감도를 확인했을 때 conventional PCR의 검출민감도는 1~10 pg이었다.

• Mycobiology
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• 제50권5호
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• pp.382-388
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• 2022

White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.

• 대한수의학회지
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• 제34권1호
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• pp.115-125
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• 1994

In this study, we try to establish the rapid and specific detection system for Salmonella species. The PhoE gene of Salmonella species was amplified with two specific primers, ST5 and ST8c, using PCR. The probe prepared from the amplified PhoE gene was sequenced and applied for Southern blot analysis. After PCR with ST5 and ST8c primers for PhoE gene, DNA bands of expected size(365bp) from 7 different Salmonella species were detected, but not from 12 enterobacteriaceae and 3 gram positive bacteria. PCR was highly sensitive to detect up to 10fg of purified DNA template and to identify Salmonella species with only 320 heat-lysed bacterial cells. The inhibition of PCR amplification from stool specimen was occurred with 50-fold dilution but disappeared over 100 fold dilution of samples. It was confirmed that the PhoE genes were amplified and cloned with over 97% nacleotide sequence homology of PCR products compared with that of S. typhfmurium LT2. The DNA probe derived from S. typhimurium TA 3,000 showed highly specific and sensitive reaction with PCR products of all tested Salmonella species. These results indicate that PCR was rapid and sensitive detection method for Salmonella species and DNA probe prepared from S. typhimurium TA 3,000 was specific to identify PCR products of different Salmonella species.