• 한국자원식물학회지
• /
• 제16권1호
• /
• pp.40-48
• /
• 2003

C. destructans는 인삼에서 가장 문제가 되고 있는 뿌리섞음병을 유발하는 매우 중요한 미생물이다. 현재까지 정상적인 인삼포장이나 폐포지에서도 이 병원균의 농도를 조사할 만한 방법이 없어 이를 쉽게 조사함으로서 인삼 예정지 관린시 도움을 줄 수 있는 새로운 방법이 절실이 요구되고 있다. 본 연구에서는 nested PCR이란 분자생물학적 방법을 이용하여 효과적으로 매우 낮은 농도의 C. destructans을 검출할 수 있는 방법을 개발하였다. 2개의 universal ITS primers(ITS5F와 ITS4R)을 사 용 하 여 Cylindrocarpon spp.의 rDNA로부터 ITS영역을 증폭하였다. 이어 C. destructans의 specific primer(Nest 1 과 Nest 2)을 사용하여 최적의 PCR조건으로 재증폭시켜 밴드를 확인하였다. 또한 이런 2번의 과정을 4개의 primer를 동시에 사용함으로서 한번에 확인할 수 있는 방법을 개발하였으며 이에 따른 PCR조건도 확립하였다. 따라서 본 방법에 의해서 인삼포장의 토양에서 채취된 매우 낮은 농도의 wild type C. destructans spore로부터 성공적으로 positive band을 확인함으로써 추후 인삼포장의 선정 및 4년생에서 6년까지(홍삼포) 재배기간등의 예측에 활용 될 것으로 생각된다.

• 원예과학기술지
• /
• 제28권4호
• /
• pp.672-677
• /
• 2010

유전자변형 장미 개발에 이용된 형질전환 벡터 pSPB130을 검출할 있는 duplex PCR이 개발되었다. 유전자변형 장미를 검출하기 위해 anthocyanin synthase($ANS$)가 장미 내 재유전자로 PCR 검사법에 이용하였다. 107bp의 PCR 산물을 얻을 수 있는 RHANS-KF/KR primer가 ANS 유전자를 증폭시키는데 이용되었고, 이것을 이용하여 9개의 다른 식물에 대해서 PCR한 결과 장미에서만 특이적인 증폭산물이 확인되었다. GMRH-KF/KR primer가 형질전환 벡터 pSPB130에 삽입된 35S promoter와 flavonoid 3',5'-hydroxylase($F3^{\prime}5^{\prime}H$) 사이의 염기서열을 확인할 수 있도록 제작되었다. 본 연구에서 개발된 duplex PCR의 검정한계치는 약 0.5%이며, 이러한 duplex PCR이 우리나라 미승인 유전자변형 장미를 모니터링 하는데 유용하게 사용될 수 있을 것이다.

• 대한의생명과학회지
• /
• 제4권2호
• /
• pp.129-135
• /
• 1998

Cytochrome P45O 2El (CYP2El)의 retropseudogene이 사람에 존재하는지 확인하기 위해 혈액에서 분리한 DNA를 주형으로 한 polymerase chain reaction (PCR)을 수행하였다. Primer는 CYP2E1 유전자를 기초하여 여러개 제작하되 적절한 조합에 따라 정상유전자와 retropseudogene이 동시에 증폭될 수 있도록 고안하였다. 본 실험의 결과 그동안 예상은 되었지만 DNA차원에서 미처확인되지 못하였던 CYP2E1의 retropseudogene을 직접 증폭해낼 수 있었다. 세부 구조는 Southern blotting과 DNA 염기서열 결정에서 최종 분석되었다. PCR 반응으로 증폭된 부분에서는 염기서열이 mRNA와 완전히 일치하고 있는 점으로 미루어서 CYP2E1의 retropseudogene은 비교적 최근에 발생했을 것으로 추정되었다.

• 한국약용작물학회지
• /
• 제18권5호
• /
• pp.329-337
• /
• 2010

'Angelicae Pubescentis Radix' (APR) is an important oriental medical preparation. In Korea, Aralia continentalis has been recognized as the source plant of APR. Aralia cordata, which is difficult to distinguish from A. continentalis, and Heracleum moellendorffii, which is frequently used in lieu of A. continentalis, are traded in Korean herbal markets. In contrast, in China, Angelica pubescens is recognized as the source plant of APR. In this study, we devised a method not only to discriminate A. contientalis from A. cordata, but also to discriminate both A. contientalis and A. cordata from H. moellendorffii and A. pubescens. Based on the discrepancy in the sequences of specific regions of ITS, we designed a Cont F/ Cont R primer set to amplify a 173 bp PCR band that appears only in A. continentalis. Additionally, we designed an Ara F/ Ara R primer set to amplify a 278 bp PCR band that appears in both A. continentalis and A. cordata. Using these primer sets and the ST R primer to confirm the PCR amplification results, we developed a simple multiplex PCR method for differentiating A. continentalis from A. cordata and to concurrently differentiate both A. continentalis and A. cordata from other APR herbs.

• 현장농수산연구지
• /
• 제5권1호
• /
• pp.57-63
• /
• 2003

The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.

• Journal of Microbiology and Biotechnology
• /
• 제17권5호
• /
• pp.806-811
• /
• 2007

Bacillus anthracis is a soil pathogen capable of causing anthrax that is closely related to several environmental species, including B. cereus, B. mycoides, and B. thuringiensis. DNA homology studies showed that B. anthracis, B. cereus, B. mycoides, and B. thuringiensis are closely related, with a high sequence homology. To establish a method to specifically detect B. anthracis in situations such as environmental contamination, we initially performed RAPD-PCR with a 10-mer random primer and confirmed the presence of specific PCR bands only in B. anthracis species. One region specific for B. anthracis was cloned and sequenced, and an internal primer set was designed to amplify a 241-bp DNA fragment within the sequenced region. The PCR system involving these specific primer sets has practical applications. Using lyses methods to prepare the samples for PCR, it was possible to quickly amplify the 241-bp DNA segment from samples containing only a few bacteria. Thus, the PCR detection method developed in this study is expected to facilitate the monitoring of environmental B. anthracis contamination.

• 한국식물병리학회지
• /
• 제14권3호
• /
• pp.223-228
• /
• 1998

Turnip mosaic virus)TuMV-Ca) was isolated from a Chinese cabbage showing severe mosaic and black necrotic spots symptoms in Korea. The virus was identified as a strain of TuMV by its host range test, particle morphology, serology, double stranded RNA analysis. For detection of the virus, reverse transcription and polymerase chain reaction(RT-PCR) was performed with a set of 18-mer TuMV-specific primers to amplify a 876 bp DNA fragment The virus was rapidly detected from total nucleic acids of virus infected tissues as well as native viral RNA of purified virion particles by RT-PCR. Detection limit of the viral RNA by RT-PCR was 10 fg.

• 한국임상수의학회지
• /
• 제15권1호
• /
• pp.140-145
• /
• 1998

The present study was carried out for the rapid and accurate detection of Anaplasma marginale in cattle using Polymerase Chain Reaction. One pair of primer, BAP-2 and AL34S, were designed to amplify a 409 Up fragment of the A marginale membrane surface protein encoding beta($msp{\beta}l$) gene with a hilly sensitive and specific PCR. A marginale isolated from naturally infected calf in Chonbuk area were used to obtain target genomic DNA for PCR. This study showed that a 409 bp of $msp{\beta}l$ gene fragment could be detected as little as 15 fg of purified A marginale genomic DNA. The amplified fragment with PCR was checked for the identification of $msp{\beta}l$ gene by enzyme restriction and sequencing. Also, the target DNA extracted directly from blood were used in the PCR reactions without prior purification to shorten the detection time. The PCR in the present study was considered convenient and rapid method for the detection of A marginale in whole blood of infected cattle.

• 대한의생명과학회지
• /
• 제9권3호
• /
• pp.139-144
• /
• 2003

Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

• 한국동물위생학회지
• /
• 제35권4호
• /
• pp.289-294
• /
• 2012

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Infections with Mycoplasma species can cause a variety of problems in living organisms and in vitro cell cultures. In this study, we investigated the usefulness of a genus-specific consensus PCR analysis method to detect Mycoplasma species. The developed consensus primer pairs MycoF and MycoR were designed specifically to amplify the 16S ribosomal RNA gene (rRNA) of Mycoplasma species by the optimized PCR system. The developed consensus PCR system effectively amplified 215 bp of Mycoplasma genus-specific region of 16S rRNA. In conclusion, we recommend this consensus PCR for monitoring Mycoplasma species in animals, human and cell culture system.