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소, 돼지, 가금육류의 신속한 동정을 위한 TaqMan<sup>ⓡ</sup> probe를 이용한 real-time PCR 개발
고바라다,김지연,나호명,박성도,김용환,Koh, Ba-Ra-Da,Kim, Ji-Yeon,Na, Ho-Myung,Park, Seong-Do,Kim, Yong-Hwan 한국동물위생학회 2012 韓國家畜衛生學會誌 Vol.35 No.3
Species-specific $TaqMan^{(R)}$ probe-based real-time PCR assays were developed for detection of beef, pork, chicken, duck, goose and turkey. The primer and probe sets used in this study were designed to be complementary to fibroblast growth factor (FGF) for cattle and pig, mitochondrial NADH dehydrogenase (ND) subunit 3 and ND2 for chicken and duck, 12S rRNA for goose and turkey, respectively. As internal positive control we used conserved region in the ribosomal 18S RNA gene to ensure the accuracy of the detection of target DNA by real-time PCR. We confirmed that real-time PCR assays with the primer and probe sets were positive for cattle, pig and chicken intended target animal species with no cross-reactivity with other non-target animal species. Only >50 ng DNA of beef show cross-reactivity in the determination of duck. Using species-specific primer and probe sets, it was possible to detect amounts of 0.1 ng DNA of cattle and pig, 1.0 pg DNA of chicken, duck and turkey, and 0.1 pg DNA of goose for raw samples, respectively. The detection limits were 0.1 ng DNA of cattle, 1.0 ng DNA of pig and 1.0 pg DNA of chicken for DNA mixtures (beef, pork and chicken) extracted from heat-treated ($121^{\circ}C$/5 min) meat samples. In conclusion, it can be suggested that the $TaqMan^{(R)}$ probe-based assay developed in this study might be a rapid and specific method for the identification of meat species in raw or cooked meat products.
바바리양에서 발생한 Streptococcus equi subsp. zooepidemicus 감염증
고바라다,박성도,김재익,박종태,Koh, Ba-Ra-Da,Park, Seong-Do,Kim, Jae-Ik,Park, Jong-Tae 대한수의학회 2007 大韓獸醫學會誌 Vol.47 No.4
An eight years old female barbary sheep (Ammotragus lervia), which bred at the Gwangju Uchi Park Zoo had shown anorexia, depression, respiratory problem for several weeks after parturition. In necropsy, extensive necrotizing pneumonia was found with severe immunocytes infiltration in the alveolar spaces and bronchioles. Pulmonary pleura were thickened with fibrin and inflammatory cells. Bacteria were isolated from lung and identified as Streptococcus equi subsp. zooepidemicus (SEZ) by biochemical tests and PCR on sodA and gusA genes, though seel gene was not detected. Isolation of zoonotic SEZ in public place such as a zoo should be emphasized for the public health mangagement.
Multiplex allele specific PCR 방법을 이용한 한우고기와 젖소고기의 신속한 판별
고바라다,Koh, Ba-Ra-Da 대한수의학회 2005 大韓獸醫學會誌 Vol.45 No.3
Here I describe a multiplex allele specific PCR-based approach for the rapid detection between Hanwoo and Holstein meat associated with Melanocortin 1 receptor (MC1R) gene. Specific and universal oligonucleotide primers were used in combination to detect the presence of a single nucleotide polymorphism within the bovine MC1R DNA sequence. The presence of the bovine MC1R gene is indicated by the production of a single control PCR product, whilst positive samples generate an alternative smaller specific product over the same region. The mutations in MC1R104 codon revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. As little as 0.39 ng and 1.56 ng of genomic DNA of Hanwoo and Holstein could be detected by MAS-PCR assay, respectively. This technique, which is widely used in human genetic screening, provides a reliable and sensitive result that has not been documented for the identification of bovine coat color. The MAS-PCR assay approach was proven to be useful in complementing routine beef DNA analysis for differentiation of these MC1R variants and it would facilitate the screening of deceiving sales of Holstein meat in the butcher shop.
돼지고기 제품 내 닭고기 검출을 위한 TaqMan<sup>®</sup> real-time PCR의 적용
고바라다,김지연,나호명,박성도,김용환,Koh, Ba-Ra-Da,Kim, Ji-Yeon,Na, Ho-Myung,Park, Seong-Do,Kim, Yong-Hwan 한국동물위생학회 2013 韓國家畜衛生學會誌 Vol.36 No.3
Many consumers are increasingly concerned about the meat they eat, and accurate labelling is important due to public health, economic and legal concerns. Meat species adulteration is a common problem in the retail markets. In this study, a TaqMan$^{(R)}$ quantitative real-time polymerase chain reaction (PCR) assay was applied for its ability to quantify chicken meat, which was not indicated on the label, in 79 commercial pork products (ham, sausages, bacon and ground meat) producted by 10 different manufacturers. The amplification efficiency was 82.05% and the square regression coefficient ($R^2$) was 0.995. PCR results showed that 38.6% of ham samples, 50.0% of sausages samples, and 50.0% of ground meat samples were contaminated with chicken residuals, while the bacon samples were not contaminated with chicken residuals. Only twelve pork products of one of the manufacturers were in accordance with indicated in their labels. The PCR assay reported in this work could be particularly useful in inspection programs to verify the food labelling of commercial processed meats and to gain consumers' trust.
광주지역 동물보호소내 유기견의 개심장사상충과 개 브루셀라병 감염 실태조사
고바라다 ( Ba Ra Da Koh ),나호명 ( Ho Myung Na ),장미선 ( Mi Sun Jang ),김지연 ( Ji Yeon Kim ),박성도 ( Seong Do Park ) 한국가축위생학회 2007 韓國家畜衛生學會誌 Vol.30 No.1
This study was conducted to investigate the prevalence of canine heartworm infections, canine brucellosis and hematologic values from 153 free roaming dogs in the area of Gwangju city from March to November 2006. Nineteen (12.4%) of 153 samples tested with modified Knott`s technique showed positive reaction for microfilariae. Polymerase chain reaction using specific primers for D immitis amplified the expected product from all samples of 19 microfilaremic canine blood samples as determined by the modified Knott`s test for microfilariae. The seasonal infection rates of microfilariae were higher in the spring season (10/19, 52.6%) than in the other seasons. The major hematological findings in microfilaremic dogs were mild leukocytosis and mild monocytosis. A total of 100 dogs randomly selected from 153 free roaming dogs were negative for canine brucellosis by serological test using immunochromatographic antibody test kit.
식육감별을 위한 미토콘드리아 12S rRNA와 16S rRNA 유전자의 종 특이적 multiplex PCR 기법 개발
고바라다 ( Ba Ra Da Koh ),김지연 ( Ji Yeon Kim ),나호명 ( Ho Myung Na ),박성도 ( Seong Do Park ),김용환 ( Yong Hwan Kim ) 한국가축위생학회 2011 韓國家畜衛生學會誌 Vol.34 No.4
Species-specific PCR assay was developed for detection of cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey using mitochondrial 12S rRNA and 16S rRNA as target genes. Also, an internal positive control was used to detect possible false negatives by using 18S rRNA gene. We designed species-specific primers with amplicon length of 190, 219, 350, 467, 241, 119, 171, 229, 111 and 268 bp for cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey respectively. The specificity of the primers was tested against the other 10 non-target animal species and a cross-reaction was not observed. We developed two multiplex PCR assays for the simultaneous identification of Korea`s major livestock species (cattle, pig, chicken and duck) and poultry species (chicken, duck, goose and turkey) from analogous samples, retaining the same specificity. The limit of detection of the multiplex PCR assay (cattle, pig, chicken and duck) ranged between 1 pg and 0.1 pg of template DNA extracts from raw meat. Applying multiplex PCR assays to DNA extracts from experimental pork/beef and pork/chicken tested raw and heat-treated (120oC for 30 min) mixtures respectively, detection limit was 0.1% level beef in pork, pork in beef and chicken in pork and 1.0% level pork in chicken. In conclusion, this assay using gel-based capillary electrophoresis would be very useful in highly sensitive and rapid identification of animal species or ingredients in minced meat and other meat products.
소 림프절에서 Mycobacterium bovis DNA의 신속 검출과 M. bovis와 M. tuberculosis 감별을 위한 real-time PCR 개발
고바라다 ( Ba Ra Da Koh ),장영부 ( Young Boo Jang ),구복경 ( Bok Kyung Ku ),조호성 ( Ho Seong Cho ),배성열 ( Seong Yeol Bae ),나호명 ( Ho Myung Na ),박성도 ( Seong Do Park ),김용환 ( Yong Hwan Kim ),문용운 ( Yong Un Mun ) 한국가축위생학회 2011 韓國家畜衛生學會誌 Vol.34 No.4
Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical- based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.
PCR-RFLP를 이용한 한우와 젖소고기의 MC1R 유전자변이 검출
고바라다 ( Ba Ra Da Koh ),김용환 ( Yong Hwan Kim ),박성도 ( Seong Do Park ),나호명 ( Ho Myung Na ),김정남 ( Jeong Nam Kim ),성창민 ( Chang Min Sung ),이삼수 ( Sam Soo Lee ) 한국가축위생학회 2005 韓國家畜衛生學會誌 Vol.28 No.3
The melanocortin 1 receptor(MC1R) encoded by the coat color extension gene(E) plays a key role in the signaling pathway of melanin synthesis. The primers for the amplification of bovine MC1R gene were designed based on a bovine MC1R gene sequence(GenBank accession no. Y19103). A size of 483bp(482bp for Hanwoo) was amplified by PCR, digested with HpaII restriction enzyme and electrophoresed in 1.5% agarose gel. When the amplified DNA product(483bp) was digested with HpaII restriction enzyme, Hanwoo meat showed a single band of 482bp, whereas two fragments of 325bp and 158bp were detected in Holstein, Angus and meat of Hanwoo/Holstein cross cow having back coat color phenotype, respectively. The results of this experiment indicate that new designed primers of bovine MC1R gene may be useful for identification of Hanwoo meat from Holstein, Black Angus and Hanwoo/Holstein cross cow meat.
Mycobacterium bovis 와 M. tuberculosis 감별을 위한 등온증폭법
고바라다 ( Ba Ra Da Koh ),김재명 ( Jae Myung Kim ),성창민 ( Chang Min Sung ),지태경 ( Tae Kyung Ji ),나호명 ( Ho Myung Na ),박성도 ( Seong Do Park ),김용환 ( Yong Hwan Kim ),김은선 ( Eun Sun Kim ) 한국가축위생학회 2013 韓國家畜衛生學會誌 Vol.36 No.2
Mycobacterium (M.) bovis, a member of the M. tuberculosis complex (MTC), is a re-emerging, zoonotic agent of bovine tuberculosis whose prevalence probably depends on variations in direct exposure to cattle and ingestion of raw milk. Accurate species differentiation of M. bovis and M. tuberculosis is needed to distinguish between human and zoonotic tuberculosis. This study successfully developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection and differentiation of M. bovis and M. tuberculosis, however showed negative reactions in eight non-tuberculous mycobacteria (NTM) samples and ten other bacterial species. Sensitivity of this assay for detection of genomic M. bovis DNA was 10 fg/μl. And this assay successfully detected M. bovis in bovine clinical specimens. In conclusion, the LAMP assay is a simple and powerful tool for rapid detection of M. bovis in both pure bacterial culture and in clinical samples.
식용란의 플루오로퀴놀론계 합성항균제의 잔류에 관한 조사
고바라다 ( Ba Ra Da Koh ),박성도 ( Seong Do Park ),장미선 ( Mi Sun Jang ),나호명 ( Ho Myung Na ),김용환 ( Yong Hwan Kim ) 한국가축위생학회 2005 韓國家畜衛生學會誌 Vol.28 No.3
This surveies were carried out to investigate the residual levels of fluoroquinolones in chicken and quail eggs by bioassay and HPLC method. The eggs of 240 samples collected from market and farm in Gwangju Metropolitan city were examined from May to December in 2003. Residual antibiotic materials were detected from 47 samples of the 240 eggs by bioassay. Of the 240 eggs assayed, ciprofloxacin, danofloxacin, norfloxacin, ofloxacin, orbifloxacin and perfloxacin were not detected but enrofloxacin was detected from 5 samples in 228 chicken eggs and 1 sample in 12 quail eggs using HPLC with fluorescence detector by multi-residue method. 2 sample eggs in 6 sample which were detected by HPLC were not positive with bioassay. The average residual concentration of enrofloxacin was 0.494 ㎎/㎏ in 6 positive samples. The highest residual concentration of enrofloxacin was 1.83 ㎎/㎏.