• 생명과학회지
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• 제15권3호
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• pp.474-478
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• 2005

본 연구는 RAPD 기법을 이용한 종 특이 marker 개발 및 이 marker의 SCAR marker로의 개발을 목표로 수행되었다. Random primer 700개에 대하여 PCR 수행결과, 품종간, 개체간에 많은 다형성이 관찰되었으며 품종특이적인 양상을 나타내는 MG30, MG53의 primer는 각각 2.0kb, 2.3kb의 위치에서 제주말과 더러브렛종의 특이적인 RAPD 단편을 나타내었다. 이들 단편들 중 품종 특이적인 단편을 클로닝한 후 random primer가 포함된 부분의 염기서 열을 결정하였다. 10 bp의 RAPD random primer에 10bp의 염기를 추가하여 SCAR primer를 제작하였다. SCAR marker의 수행결과 RAPD marker와 같은 2.3kb, 2.0kb의 크기에서 제주마와 더러브렛종에 특이적인 하나의 밴드가 증폭되었다. 따라서 이 Cnh-SCAR marker는 보다 안정적이고 재현성 있는 marker로서 사용이 가능하여 제주말의 판별에 유용하게 사용될 수 있을 것이다.

• Mycobiology
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• 제37권4호
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• pp.317-319
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• 2009

In this study, in an effort to develop a method for the molecular detection of Tricholoma matsutake in Korea from other closely related Tricholomataceae, a species-specific PCR primer pair, TmF and TmR, was designed using nuclear ribosomal intertranscribed spacer (ITS) sequences. The DTmF and DTmR sequences were 5'-CCTGACGCCAATCTTTTCA-3' and 5'- GGAGAGCAGACTTGTGAGCA-3', respectively. The PCR primers reliably amplified only the ITS sequences of T. matsutake, and not those of other species used in this study.

• Food Science and Biotechnology
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• 제17권2호
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• pp.357-361
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• 2008

One of the major allergenic proteins in common buckwheat (Fagopyrum elculentum) was found to be a BW10KD. In this work, allergenic BW10KD genomic DNAs from the native common buckwheat 'Pyeongchang' and Tartarian buckwheat 'Clfa47' were cloned by polymerase chain reaction (PCR), and their nucleotide sequences were determined. In addition, a novel PCR assay targeting the allergenic BW10KD gene was developed to detect and differentiate both buckwheat species in food. The nucleotide sequences of the BW10KD genomic DNA from 'Pyeongchang' and 'Clfa47' were 94% identical. Base differences in the nucleotide sequences of the BW10KD genes are probably useful as a molecular marker for species-specific identification. The 'Pyeongchang'-specific primer set 154PF/400PR and the 'Clfa47'-specific primer set 154DF/253DR generated 247 and 100 bp fragments in singleplex PCR, respectively. A duplex PCR assay with 2 species-specific primer sets simultaneously differentiated the 'Pyeongchang' and 'Clfa47' in a single reaction. The PCR assay also successfully allowed for the rapid detection of buckwheat ingredients in foods.

• 산업기술연구
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• 제28권A호
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• 한국산림과학회지
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• 제96권5호
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• pp.585-590
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• 2007

Rhizina undulata is the fungus, which causes Rhizina root rot on coniferous trees. Nested-PCR using ITS-specific primer was applied to detect R. undulata from the soils of Japanese black pine (Pinus thunbergil) forests infested with the disease in Seocheon, Chungnam Province, South Korea. Soil samples were collected from four different sites, both dead trees and fruit bodies of R. undulata were present, dead trees only present, fruit bodies only present, and both were absent. Nested-PCR products specific to R. undulata ITS-region were amplified. Positive reactions were found in some samples from the sites, where dead trees and fruit bodies of R. undulata were absent as well as where both of those were present. R. undulata was mainly detected in the soil samples from the depth of 5~20 cm under the soil surface. These results show that the nested-PCR could be used to diagnose the presence or potential infestation of R. undulata in the soils of pine forests.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.737-744
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• 2004

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Vairimorpha spp. and Nosema spp. and identification of Vairimorpha necatrix from Lepidoptera insects. Three sets of primers were selected from different genomic sequences to specifically amplify an 831 bp amplicon within the SSU rRNA gene, specific for both Vairimorpha spp. and Nosema spp. (MSSR primer); a 542 bp amplicon within the SSU rRNA gene, specific for Vairimorpha spp. (VSSU primer); and a 476 bp amplicon within the actin gene, specific for Vairimorpha necatrix (VNAG primer). Using the primers in conjunction with multiplex PCR, it was possible to detect Vairimorpha spp. and Nosema spp. and to differentiate between them. The sensitivity of this PCR assay was approximately 10 spores per milliliter. It is proposed that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting Vairimorpha spp. and Nosema spp. in Lepidoptera insect.

• The Plant Pathology Journal
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• 제15권5호
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• pp.287-294
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• 1999

A technique based on the polymerase chain reaction (PCR) for the specific detection of genus Phytophthora and Phytophthora cryptogea-P. drechsleri complex group was developed using nucleotide sequence information of ribosomal DNA (rDNA) regions. The internal transcribed spacers (ITS) including 5.8S were sequenced for P. cryptogea-P. drechsleri complex group and its related species. Two pairs of oligonucleotide primers were designed. Primer pair ITS1/Phy amplified ca. 240 bp fragment in 12 out of 13 specie of Phytophthora, but not in Pythium spp., Fusarium spp.and Rhizoctonia solani. Primer pair rPhy/Pcd amplified 549 bp fragment only in P. cryptogea-P. drechsleri complex group, but not in other Phytophthora spp.and other genera. Specific PCR amplification using the primers was successful in detecting Phytophthora and P. cryptogea-P. drechsleri complex group in diseased plants.

• 식물조직배양학회지
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• 제25권5호
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• pp.309-313
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• 1998

본 연구는 국내의 경우 제주도의 일부지역에서만 자생하는 흑오미자(Schisandra nigra)를 RAPD법을 이용하여 자웅동주 및 자웅이주 식물의 특이적 Marker를 탐색하고저 실시 하였다. 10-mer로 구성된 80종류의 random primer를 사용하여 흑오미자를 분석한 결과 기존의 재배되고 있는 오미자(Schisandra chinensis) 또는 남오미자(Kadsura japonica)와는 다른 band pattern을 보여 주었다. 흑오미자의 자웅동주. 암그루, 숫그루의 3품종을 상기와 동일하게 80개의 primer를 사용하여 RAPD를 분석한 결과, 5종류의 random primer(OPA-17, OPA-19, OPB-3, OPB-9, OPB-16)에 대해서는 각 품종에 대한 특이적인 band가 검출되었다. 숫그루, 암그루 식물과는 다르게 자웅동주 식물은 3개체(1호 2호, 3호)간에도 서로 다른 band pattern을 보이는 특징을 갖고있다. 숫그루 특이적인 band pattern은 OPB-3 primer을 이용할 경우 750bp에서 검출되었고, 암그루는 OPA-19 primer에서 950bp, 1690bp와 OPB-3 primer의 경우 700bp에서 자웅동주 및 숫그루에서 나타나지 않는 band가 검출되었다. 이러한 결과는 흑오미자의 숫그루 및 암그루에서 나타난 특이적인 band가 유묘시기에 암ㆍ수 개체를 조기에 구별하는 genetic marker로 사용할 수 있으리라 기대된다.

• 미생물학회지
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• 제43권3호
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• pp.179-185
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• 2007

본 연구는 polymerase chanin reaction(PCR)기법과 DNA enzyme-linked immunosorbent assay(DNA ELISA) 기법을 이용하여 토양내 식물병원균인 Ralstonia solanacearum를 검출하고자 하였다. 토양 시료로부터 분석에 사용될 R. solanacearum DNA를 추출하기 위하여 몇 가지 방법을 비교 평가한 결과 기존의 DNA 추출 방법에 비하여 Guanidin isothiocyanate와 Chelex-100 resin을 사용하는 방법 이 토양 내에 존재하는 다양한 중류의 반응 저해 물질과 R. solanacearum만의 고유한 PCR반응 저해물질들을 제거하는 데에 효과적이었다. R. solanacearum만을 특이적으로 검출하기 위해 fliC유전자 부위에 특이적인 몇 종의 primer들을 제작하였다. 이들 중 높은 민감도와 특이도를 나타내는 두 set의 primer RsolfliC(forward; 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.)와 RS_247 (forward; 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3, designed by this study)를 선정하여 nested PCR을 수행할 수 있도록 고안하였다. Nested PCR primer에 biotin을 표지하였고 nested PCR산물의 내부 서열과 특이적으로 교잡반응을 할 수 있는 probe를 제작하여 PCR 결과를 DNA-EIA반응으로 확인 분석할 수 있도록 하였다. Primary PCR과 nested PCR의 산물을 전기영동 상에서 확인한 결과, nested PCR이 약 $10^2$정도의 높은 민감도를 나타내었고 DNA-EIA의 경우 $10^2P{\sim}10^3$정도의 민감도를 상승시켜주는 것으로 확인되었다.

• Applied Biological Chemistry
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• 제49권3호
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• pp.192-195
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• 2006

국내에서 개발된 비타민 E 강화 유전자변형 들깨의 정성 PCR 분석법의 개발을 위해 들깨의 내재 유전자로써 KAS-I (Beta-ketoacyl-ACP synthase I)를 선별하였고, 이러한 내재유전자를 특이적으로 증폭시킬 수 있는Primer(Pfru3-F/R)쌍을 이용한 PCR에서 95 bp의 PCR증폭 산물을 얻었으며, 들깨를 포함한 16개 작물에 대해 PCR을 수행한 결과에서 들깨만이 특이적으로 증폭되는 것을 확인하였다. 또한, 비타민 E 강화 유전자변형 들깨에 삽입된 TMT(${\gamma}$-tocopherol methyltransferase) 유전자와 OCS(Octopine synthase) terminator 연결 부위를 증폭시켜 148 bp의 PCR 산물을 얻을 수 있는 primer(TMTO-F/R)를 제작하였으며, 이러한 두 쌍의 primer를 이용하여 국내 개발된 비타민 E 강화 유전자변형 들깨의 PCR 정성 분석법을 확립하였다.