• 생명과학회지
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• 제9권6호
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• pp.671-676
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• 1999

국내에서 재배되는 7종의 마늘과 오국종인 헝가리종 및 중국 산동종을 수집하여 총 70종의 임의 primer를 이용하요 RAPD분석을 실시한 결과 32개의 primer에서 종간에 다형성(polymorphism)을 보이는 DNA벤드를 확인하였다. PCR에 의해 증폭된 DNA밴드 수는 151개였으며 그 중 125개의 밴드가 수집종 간에 다형성을 보였다. 다형성을 보인 밴드를 대상으로 집단분석을 실시한 결과 유전적 거리가 0.271인 값에서 9종의 수집마늘은 두 개의 group으로 나누어 졌는데, Group I은 창녕종과 헝가리종 이었고 Group II는 남도, 산동, 예천, 의성, 정선, 영월 및 단양종으로 분류되었다. 마늘의 주요 생태형인 난지형과 한지형은 유전적 거리가 0.200값을 전후하여 나누어 졌다. 각각의 primer로부터 나타난 밴드들은 지방종 간에 특이성을 보이는 것이 다수 존재하여 외국종과 국내종 마늘이나 국내종 간의 종을 구분하는 표식이자로 사용할 수 있을 것이다.

• Parasites, Hosts and Diseases
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• 제37권4호
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• pp.257-263
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• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

• Plant Biotechnology Reports
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• 제4권1호
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• pp.49-52
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• 2010

A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.

• 대한수의학회지
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• 제39권2호
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• pp.343-352
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• 1999

Vancomycin resistant enterococci (VRE) have emerged as an important nosocomial pathogen. Since 1989 the Center for Disease Control, United States, has reported a rapid increase in the incidence of enterococcal bacteremia and endocarditis infection by VRE. It was suggested that the use of avoparcin was associated with the appearance of VRE in animal husbandry. To date, several detection methods have been used based on conventional methods of culture and gene detection. However, these methods have some limitations such as time-consuming, laborious and additional differential needs. Therefore, In this study a multiplex PCR method was established to detect and differentiate resistance types of enterococci which specifically amplify the four van genes encoding vancomycin resistance elements. Using the method, we investigated the incidence rates and types of VRE from farms using or not using avoparcin. A total of 1091 animal fecal samples were collected from 70 pig and 32 poultry farms. A total of 425 of enterococci were isolated from samples. Of the 425 isolates, 11 of the them showed a pattern of high-level vancomycin resistance (MIC : $64{\sim}256{\mu}g/ml$) which was associated with the presence of the vanA or vanB gene. Fifty-seven isolates showed a pattern of low-level vancomycin resistance (MIC : $3{\sim}8{\mu}g/ml$) associated with the vanC-1 or vanC-2 gene. Interestingly, all isolates with high-level vancomycin resistance were from farms that have never used avoparcin. Moreover, the high-level VRE isolation rate in Korea (2.58%) was much lower than that of other countries (50% in England, 7% in Belgium) where avoparcin have been used. In conclusion, the multiplex PCR method established in this study could be applied for detection of VRE.

• 약학회지
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• 제49권3호
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• Journal of Microbiology
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• 제42권4호
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• pp.365-368
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• 2004

The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA $\rightarrow$CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gin to Arg.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.113.2-114
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• 2003

Five pepper-infecting potyviruses, Pepper mottle virus (PepMoV), Chilli veinal mottle virus (CVMV), Pepper veinal mottle virus (PVMV), Pepper severe mosaic virus (PSMV) and Tobacco each virus (TEV), are known filamentous virus and can be infected pepper crops systemically. To understand pathology and genome information of the five viruses on pepper plants, host reactions and sequences were compared to the 5 viruses. Five potyviruses were inoculated onto some typical cultivars of hot peppers and compared their symptoms, and virus accumulations. A set of degenerate primers for potyviruses were applied to 5 viruses and RT-PCR was performed. RT-PCR products containing partial nuclear inclusion b and coat protein (CP) genes were cloned. Then, oligo dT primer and species-specific primer were redesigned to amplify the C-terminal part of CP and 3' noncoding regions of each viruses. Sequences of the viruses were analyzed and compared to serological relationships among the viruses. The data can be useful for screening of potyviruses in pepper plants and pathogen-derived transgenic pepper plant development.

• ALGAE
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• 제28권1호
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• pp.31-43
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• 2013

This review provides a comprehensive summary of the PCR primers and profiles currently in use in our laboratory for red algal DNA barcoding and phylogenetic research. While work focuses on florideophyte taxa, many of the markers have been applied successfully to the Bangiales, as well as other lineages previously assigned to the Bangiophyceae sensu lato. All of the primers currently in use with their respective amplification profiles and strategies are provided, which can include full fragment, overlapping fragments and what might best be called "informed overlapping fragments", i.e., a fragment for a marker is amplified and sequenced for a taxon and those sequence data are then used to identify the best primers to amplify the remaining fragment(s) for that marker. We extend this strategy for the more variable markers with sequence from the external PCR primers used to "inform" the selection of internal sequencing primers. This summary will hopefully serve as a useful resource to systematists in the red algal community.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2001년도 추계학술대회
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• pp.146-156
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• 2001

The biosynthetic gene clusters for bluensomycin and spectinomycin were isolated and characterized from the bluensomycin producer, Streptomyces bluensis ATCC27420 and the spectinomycin producer, Streptomyces spectabilis ATCC27741, respectively. PCR primers were designed specifically to amplify a segment of dTDP-glucose synthase gene based on its conserved sequences of several actinomycete strains. By screening cosmid libraries using amplified PCR fragments, 30-kb and 45-kb DNA fragments were isolated from Streptomyces bluensis and Streptomyces spectabilis, respectively. Sequencing analysis of them revealed that each contains 15 open reading frames (ORFs). Some of these ORFs were turned out to be antibiotic resistance genes (blmA and speN), dTDP-glucose synthase genes (blmD and spcD), and dTDP-D-glucose 4,6-dehydratase genes (blmE and spcE), suggesting that the blm and spec gene clusters are likely involved in the biosynthesis of bluensomycin and spectinomycin, respectively.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.732-739
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• 2014

We describe the development of a polymerase chain reaction method for the rapid, precise, and specific detection of the Xanthomonas oryzae pv. oryzae (Xoo) K3a race, the bacterial blight pathogen of rice. The specific primer set was designed to amplify a genomic locus derived from an amplified fragment length polymorphism specific for the K3a race. The 1,024 bp amplicon was generated from the DNA of 13 isolates of Xoo K3a races out of 119 isolates of other races, pathovars, and Xanthomonas species. The assay does not require isolated bacterial cells or DNA extraction. Moreover, the pathogen was quickly detected in rice leaf 2 days after inoculation with bacteria and at a distance of 8 cm from the rice leaf 5 days later. The results suggest that this PCR-based assay will be a useful and powerful tool for the detection and identification of the Xoo K3a race in rice plants as well as for early diagnosis of infection in paddy fields.