• 대한수의학회지
• /
• 제44권3호
• /
• pp.399-405
• /
• 2004

Haemophilus somnus, Mycoplasma bovis and Pasteurella multocida were responsible for respiratory diseases in bovine. Methods for identifying these bacteria had poor sensitivity and specificity. In this paper, PCR assays were applied for rapid identification of H. somnus, M. bovis, P. multocida B:2 and P. multocida capsular types. The specific PCR products were amplified from H. somnus, but not from other bacteria. Ten-fold diluted H. somnus were mixed with P. multocida and then the mixed cultures were inoculated on agar plates. After incubation, PCR was performed with harvest from agar plates and could detect as few as 3.4 CFU/ml of H. somnus. The primers MboF and MboR produced an amplification product unique to M. bovis and sensitivity of PCR was as low as 100 pg of DNA. Only serotype B:2 of P. multocida, the causal agent of haemorrhagic septicemia in bovine, was specifically amplified in PCR among the 16 reference serotypes. The multiplex capsular PCR typing for P. multocida was produced the P. multocida specific product as well as the capsular serogroup-specific product. The present PCR assays should be useful for the rapid identification of bacterial pathogens from bovine respiratory diseases.

• The Plant Pathology Journal
• /
• 제19권3호
• /
• pp.159-161
• /
• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• 대한약침학회지
• /
• 제12권1호
• /
• pp.13-20
• /
• 2009

Objective : This study developed species-specific PCR and PCR-RFLP to detect the adulteration of Fel ursi products with cattle and pig bile juices. Methods : All the primers for PCR and PCR-RFLP in this study were designed based on nucleotide sequences of cytochrome b genes in the mitochondria. Results : The species-specific PCR amplified a DNA fragment of 214, 214, 295, and 167 bp from Fel ursi product, bear fur, cattle bile juice, and pig bile juice, respectively. The survey using the speciesspecific PCR indicated that some of commercial Fel ursi products were adulterated with cattle and pig bile juices. PCR-RFLP using the restriction endonucleases, HaeIII and HinfI enabled differentiation among Fel ursi product, cattle bile juice, and pig bile juice. Bear furs from two animals showed variations in PCR-RFLP patterns with HaeIII. Discussion : The detection methods of the species-specific PCR and PCR-RFLP could be useful in eliminating adulterated Fel ursi products from the market.

• Preventive Nutrition and Food Science
• /
• 제3권2호
• /
• pp.198-202
• /
• 1998

The arbitrary primed polymerase chain reaction (AP-PCR) has been used to detect the genetic alternations in the related species. Simple and reproducible fingerprints of complex genomes can be generated using single arbitrary chosen primers and the PCR. The technique was applied to the Oryza species and characterized the relationship among three cultivars of rice species based on theresult of genomic DNA fingerprints. The results indicated that the polymorphism revealed in rice strains and the differences in the PCR product pattern could be represented for each strainis. There was many variationsin the PCR product pattern between cv. Dongin(japonica type)and cv.Hyangdo (indica type), and our chosen AP-primers can ge as markers for strain identification and verfication.

• Journal of Plant Biology
• /
• 제38권3호
• /
• pp.267-274
• /
• 1995

Based upon the nucleotide sequence of As strain of cucumber mosaic virus (CMV-As0 RNA4, coat protein (CP) gene was selected for the design of oligonucleotide primers of polymerase chain reaction (PCR) for detection and identification of the virus. Reverse transcription and polymerase chain reaction (RT-PCR) was performed with a set of 18-mer CMV CP-specific primers to amplify a 671 bp fragment from crude nucleic acid extracts of virus-infected leaf tissues as well as purified viral RNAs. The minimum concentrations of template viral RNA and crude nucleic acids from infected tobacco tissue required to detect the virus were 1.0 fg and 1:65,536 (w/v), respectively. No PCR product was obtained when potato virus Y-VN RNA or extracts of healthy plants were used as templates in RT-PCR using the same primers. The RT-PCR detected CMV-Y strain as well as CMV-As strain. Restriction analysis of the two individual PCR amplified DNA fragments from CMV-As and CMV-Y strains showed distinct polymorphic patterns. PCR product from CMV-As has a single recognition site for EcoRI and EcoRV, respectively, and the product from CMV-Y has no site for EcoRI or EcoRV but only one site for HindIII. The RT-PCR was able to detect the virus in the tissues of infected pepper, tomato and Chinese cabbage plants.

• 한국수정란이식학회지
• /
• 제15권2호
• /
• pp.175-180
• /
• 2000

The possible use of micromanipulative biopsy and PCR of the biopsied embryonic cells was tested to produce sexed bovine embryos in practical terms. By micromanipulation and PCR techniques, higher survival rate and accurate sexing of demi-embryos were btained. Bovine oocytes matured and fertilized in vitro were co-cultured with bovine oviductal epithelial cell (BOEC) monolayer in USU-6 medium supplemented with 15% FBS, and the embryos of 37% (327/885) were developed to blastocysts. Among 111 blastocysts produced by invitro, only 7 (6.3%) embryos were found unable to determine their sex, probably due to the loss of cells, since no PCR product was found from those cells. All the remaining 104 (93.7%) demi-embryos survived micromanipulation and demonstrated male-specific product or bovine-specific product alone suggesting that correct sexing of the sample. Forty-three point one percent(25/58) of manipulated and cryopreserved demi-embryos after thawing were survived. Final verification of the sexed embryos is necessary to make sure the same sex in fetus and newborn calf upon embryo transfer. The established sexing method on a large number of bovine embryos from previous and this study suggests that this a could be used practically in the field.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제7권4호
• /
• pp.212-215
• /
• 2002

Tyrosinase transcript In the blood Is known as the marker of malignant melanoma and it has been often determined by using reverse transcription-polymerase chain reaction (RT-PCA) . However, after the PCR process, the quantification of amplified CDMA by the gel electrophoresis is not reliable and time-consuming. for this reason, we tried to quantify the PCR product using a cuvette-type biosensor, where the oligonucleotide probe was immobilized on the cuvette surface and the single strand CDMA, the denatured PCH product, was then hybridized onto the immobilized probe to give a response signal. The response was Immediate and takes 15 min to obtain a stable signal. The biosensor was much more sensitive comparing to the gel electrophoresis method. The quantification of PCR product using a cuvette-type biosensor was feasible and rapid.

• 환경생물
• /
• 제22권2호
• /
• pp.329-335
• /
• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• 대한인간공학회지
• /
• 제19권3호
• /
• pp.23-37
• /
• 2000

Usability is one of the most important factors that affect customers' decision to purchase a product. Several studies have been conducted to model the relationship between the product design variables and the product usability. Since there could be hundreds of design variables to be considered in the model, a variable screening method is required. Traditional variable screening methods are based on expert opinions (Expert screening) in most Kansei engineering studies. Suggested in this study are statistical methods for screening important design variables by using the principal component regression(PCR), cluster analysis, and partial least squares(PLS) method. Product variables with high effect (PCR screening and PLS screening) or representative variables (Cluster screening) can be used to model the usability. Proposed variable screening methods are used to model the usability for 36 audio/visual products. The three analysis methods (PCR, Cluster, and PLS) show better model performance than the Expert screening in terms of $R^2$, the number of variables in the model, and PRESS. It is expected that these methods can be used for screening the product design variables efficiently.

• Applied Biological Chemistry
• /
• 제41권1호
• /
• pp.42-46
• /
• 1998

본 연구는 polymerase chain reaction (PCR)와 Southern hybridization을 이용하여 서로 붙어있는 나무가 한 나무인지 두 나무인지 규명하였다. 공시된 수종은 구지뽕나무와 무궁화이다. 구지뽕나무는 경상북도 성주군 금수면 봉두리 안새출 금수식당 앞에 있는 나무로 두 사람이 서로 안고 있는 형상인데, 한 나무의 두 가지인지 가까이 자란 두 나무가 붙었는지를 구별하기 어려운 나무였다. 다섯 종류의 PCR primer $(17{\sim}24-mers)$ 중 355 primer의 경우 한가지의 잎 DNA에서만 PCR 산물이 검출되어 이것을 방사선표지한 후 genomic Southern hybridization을 행하였던 바 이 probe와 결합하는 DNA 단편이 동일한 위치에서 검출되었으나 정도는 다르게 나타났다. 아울러 OPERON 10-mer kits A에 있는 20종류의 primer를 이용하여 PCR을 행한 결과 OPA01 primer (CAGGCCCTTC)에 의한 PCR 생성물은 동일한 위치의 band 뿐만 아니라 추가로 4개가 한가지의 잎 DNA에서 더 나타났다. 따라서 구지뽕나무는 두 나무가 연결되어 한 나무를 이루는 것으로 짐작된다. 또한 무궁화는 경상남도 합천군 청덕면 두곡리 청덕초등학교 내에 있는데 수령 50년 이상으로 멀리서 보면 한 나무의 형상을 하고 있으나, 가까이 다가가서 줄기의 아래부분을 보면 세 줄기가 붙어있는 형상을 하고 있다. 따라서 이 무궁화 역시 한 나무의 세 가지인지 세나무가 가까이 자라 붙었는지 PCR과 Southern hybridization 방법을 이용하여 규명하려 하였다. Southern hybridization 결과에 의하면 구지뽕나무의 분석에 사용한 probe와 결합하는 DNA 단편은 검출되지 많았으며, 35S primer에 의한 PCR 생성물은 동일하였으나 OPA04와 OPA13 primer의 경우 약간의 상이성을 보였다. 이러한 결과에 따라 무궁화는 한 나무의 세 가지인 듯하나 추가적인 연구가 필요하다고 판단된다.