• Korean Journal of Plant Resources
• /
• v.14 no.3
• /
• pp.235-240
• /
• 2001

Polymerase chain reaction (PCR) is used in a wide array of researches in plant molecular genetics and breeding. However, considerable time and cost are still required for the preparation of DNA suitable for reliable PCR results, especially in plant species containing high amounts of polyphenols. To reduce time and effort for PCR-based analysis, a simplified but reliable method was developed by a combinational employment of a simple and fast DNA extraction procedure and BLOTTO (Bovine Lacto Transfer Technique Optimizer) in reaction mixture. Genomic DNAs prepared by one-step extraction method from recalcitrant plant species such as Rubus coreanus, apple, grape and lettuce were successfully amplified by random primers in the reaction mixture containing 2 to 4% BLOTTO. Successful amplification of ${\gamma}$-TMT transgene in lettuce transformants by the specific primers was also achieved in the same condition, making rapid screening of positive transformants possible. Our results suggest that use of a simple DNA extraction procedure and incorporation of BLOTTO in reaction mixture in combination can reduce time and effort required for the analyses of a large number of germplasms and transformants by PCR-based techniques.

• Proceedings of the Korean Society of Fisheries Technology Conference
• /
• 1995.06a
• /
• pp.242-243
• /
• 1995

• Korean Journal of Plant Resources
• /
• v.16 no.1
• /
• pp.40-48
• /
• 2003

Cylindrocarpon destructans is the major pathogen inducing the root rot disease in ginseng. Up to now, there is no reliable and convenient method to analyze the spore density or population of this pathogen in ginseng-growing soil or any contaminated farmlands. Therefore, it will be very valuable to develop a new and reliable method in detecting the spore of this pathogen. In this study, a molecular biological technique using two step nested PCR method, was developed. Two universal ITS primers, ITS5F and ITS4R were used in the first round of PCR to amplify a fragment of ITS region from the genomic DNA of C. destructans. The specific prmers Nest 1 and Nest 2 were designed and used in the second round of PCR to amplify a inner fragment from the first round PCR product of C. destructans. C. destructans spore, only soil samples from the diseased ginseng farm produced the positive bands, suggesting its usefulness in detecting the C. destructans spores in soil samples. Thus it is recommended to first extract the whole genomic DNA from soil samples and use it for the PCR reaction, thereby eliminating the inhibitory activity of soil components.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.29 no.6
• /
• pp.1016-1024
• /
• 2000

The study was conducted to develop a rapid method for the detection of Listeria monocytogenes in foods via polymerase chain reaction (PCR) technique using hemolysin gene (hlyA) primers. Specificity and sensitivity of PCR, optimal conditions for PCR and application of hlyA gene primers for the detection of L. monocytogenes from milk and beef were investigeted. Each of the 20 L. monocytogenes strains gave a single 713 bp band, but other Listeria sup. and other bacteria did not show any bands. As few as 1 pg of L. monocytogenes DNA or 2.4$\times$10$^4$L. monocytogenes cells could be detected with hlyA gene primers. PCR product was most improved at 20~30 cycle in terms of removal of tailing and sensitivity. Also, the sensitivity was significantly improved by the further 10~15 cycle after 20 cycle PCR amplication. Milk (10 mL) and beef (10 g) samples were inoculated with L. monocytogenes at the concentrations ranging from 0 to 10$^{7}$ CFU/mL or g to determine the best sensitivity of PCR for the rapid detection of L. monocytogenes. PCR assay could detect 2 cells in milk with repeating PCR amplication and 2.6$\times$10$^2$cells in beef sample after 24 hr enrichment growth at 35$^{\circ}C$ in LEB.

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.7
• /
• pp.1046-1048
• /
• 2003

A polymerase chain reaction (PCR) assay was developed to differentiate buffalo meat from the meat of Ceylon spotted deer (Axis axis ceylonensis), Ceylon sambhur (Cervus unicolor unicolor), cattle (Bovine), goat (Caprine), pig (Porcine), and sheep (Ovine). A set of primers were designed according to the sequence of the mitochondrial cytochrome b gene of bubalus bubalis and by PCR amplification a band of approximately 242 bp band was observed with buffalo DNA. These primers did not cross-react with DNA of other animal species tested in the study under the specified reaction conditions. A band of 649 bp was observed for all animal species tested when DNA was amplified with the universal primers indicating the presence of mitochondrial DNA in the samples. The technique was sensitive enough to identify rotten (10 days post slaughter), dried and cooked buffalo meat. The absence of a cross reaction with human DNA using the buffalo specific primers eliminates possible false positive reactions.

• Journal of Periodontal and Implant Science
• /
• v.29 no.2
• /
• pp.349-355
• /
• 1999

The present study has been performed to develop a PCR technology to identify human immunoglobulin(Ig) allotypes with restriction fragment length polymorphism(RFLP) using a probe. Genomic DNA were ampilified with PCR tecnology using primers from peripheral blood lymphocytes of 10 periodontal patiens, whose Ig allotypes have been pre-determined by serological tecnique using heagglutination technique. The result indicated that the RFLP patterns could successfully differentiate the Ig allotypes, which suggests that this technology can be developed as a tool useful for population genetics studies.

• Journal of Veterinary Clinics
• /
• v.25 no.3
• /
• pp.211-214
• /
• 2008

This report describes a case of severe and prolonged dermatophytosis in a hedgehog that was diagnosed by PCR-RFLP, a rapid and usefulness technique for identification of many causative agents and hereditary characters. A 5-month-old female hedgehog was presented with grade 2 facial pruritus, scaling, encrustation and hemorrhage. Cytology of exudates on the face showed a suspected fungal infection. A culture and tape imprint test of the cultured colony showed many hyphae and microcornidia, suspected to belong to the Trichophyton species. In the PCR-RFLP with MvaI and Hinf I, Trichophyton mentagrophytes var. erinacei was finally identified as a causative agent. The patient completely recovered after application of nystatin cream for 17 days.

• Korean Journal of Microbiology
• /
• v.39 no.1
• /
• pp.40-44
• /
• 2003

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. In order to develop a DNA marker specific for Bacillus anthracis and to discriminate this species from Bacillus cereus group, we applied the randomly amplified polymorphic DNA (RAPD)-PCR technique to a collection of 29 strains of the genus Bacillus, including 22 species of the B. cereus group. A 709-bp RAPD marker (KHT5) specific for B. anthracis was obtained from B. anthracis BAK. The PCR product of internal primer set from the KHT5 fragment distinguished B. anthracis from the other species of the B. cereus group.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.4
• /
• pp.668-672
• /
• 2007

A microcosmal experiment using a metagenomic technique was designed to assess the effect of BTEX (benzene, toluene, ethylbenzene, and xylenes) on an indigenous bacterial community in a Daejeon forest soil. A compositional shift of bacterial groups in an artificial BTEX-contaminated soil was examined by the 16S rDNA PCR-DGGE method. Phylogenetic analysis of 16S rDNAs in the dominant DGGE bands showed that the number of Actinobacteria and Bacillus populations increased. To confirm these observations, we performed PCR to amplify the 23S rDNA and 16S rDNA against the sample metagenome using Actinobacteria-targeting and Bacilli-specific primer sets, respectively. The result further confirmed that a bacterial community containing Actinobacteria and Bacillus was affected by BTEX.

• Journal of Embryo Transfer
• /
• v.11 no.2
• /
• pp.145-150
• /
• 1996

This study was conducted to detect the Y-specific DNA in the blood of the female calf in bovine heterosexual production. Genomic DNAs of the freemartin were isolated from the blood and amplified with Y-chromosome specific DNA primer(l4lbp). In order to estimate the lower limit for the detection of XY cells, blood from a hull was diluted in cow blood to 0.01%. DNA sequencing on the PCR products was shown the same sequences as Y chromosome DNA of the normal cows. The Y specific DNA hand by PCR was detected all blood of female calf suspected to have bovine freemar tin syndrom and the karyotyping with freemartin blood was identified as XX / XY chimerism. Therefore, the PGR methods used in this study was very useful technique for the detection of freemartin in Ranwoo and Holstein.