• KSBB Journal
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• 제29권5호
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• pp.361-371
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• 2014

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.

• 미생물학회지
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• 제41권4호
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• pp.269-274
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• 2005

동물용 생 바이러스 백신 내에 mycoplasma를 검출하기 위해 polymerase chain reaction (PCR)기법과 2가지의 상품화된 PCR 검출킷트를 평가하였다. PCR기법은 시험에 사용된 모든 mycoplasma를 특이적으로 검출할 수 있었으나, 2가지의 상품화된 PCR 검출킷트는 일부의 mycoplasma를 검출하지 못하였다. 또한, PCR기법의 검출 특이도는 조류 유래 mycoplasma에 속한 4주의 표준주 및 7주의 야외분리주를 모두 검출할 수 있었다. PCR기법의 민감도는 9 CFR Mycoplasma액체배지에서 배양한 Mycoplasma 속균 및 Acholeplasma속균에 대해 $1\~100$ colony forming units/ml까지 검출할 수 있었다. 동물용생 바이러스 백신에 대해 PCR기법의 적용가능성을 평가하기 위해, 돼지 전염성위장염 및 로타바이러스 흔합백신과 개 파보바이러스 백신내에 A. laidlawii를 인공적으로 접종한 후, PCR기법의 민감도를 조사하였을 때 배양액을 이용한 검출한계와 유사하였다. 본 연구에서 사용된 PCR 기법은 동물용 생 바이러스 백신내의 mycoplasma를 신속하고 민감하게 검출할 수 있을 것으로 판단되었다.

• 한국동물위생학회지
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• 제37권4호
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• pp.247-252
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• 2014

The economic impact of swine mycoplasma infection is high. An accurate diagnosis is often difficult and time consuming. We report the development and validation of an effective multiplex polymerase chain reaction (PCR) assay that detects Mycoplasma (M.) hyopneumoniae and M. hyorhinis. The multi detection of M. hyopneumoniae and M. hyorhinis primer set were employed to detect mycoplasma species and typing of the species was performed on the basis of sequence analysis of the PCR product. The target nucleic acid fragments were specifically amplified by M. hyopneumoniae and M. hyorhinis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were used to evaluate the specificity of the multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. hyopneumoniae and M. hyorhinis.

• 대한수의학회지
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• 제39권1호
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• pp.90-95
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• 1999

A species-specific 760 base pair(bp) BamHI to EcoRI DNA fragment(fMG-2) of lipoprotein gene was isolated from a Mycoplasma gallisepticum(M gallisepticum) genomic library. Based on the DNA sequence data of fMG-2, a pair of 25bp primers was synthesized. When used in the polymerase chain reaction(PCR), 732bp DNA products were amplified from 6 standard strains and 10 field isolates of M gallisepticum, but not from 2 Mycoplasma synoviae and 7 other Mycoplasma species. The lower detection limit was 100fg of the genomic DNA. Identity of the PCR products was confirmed by comparison of patterns of restriction endonuclease analysis with AseI, DraI, EcoRV and SspI.

• 한국환경농학회:학술대회논문집
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• 한국환경농학회 2009년도 정기총회 및 국제심포지엄
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• pp.187-199
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• 2009

We present real.time, rapid detection of Mycoplasma pneumonia in phosphate buffered saline (PBS) inside a Y.channel polydimethylsiloxane (PDMS) microfluidic device by means of optical fiber monitoring of latex immunoagglutination. The latex immunoagglutination assay was performed with serially diluted Mycoplasma pneumonia solutions using highly carboxylated polystyrene particles of 390nm and 500nm diameter conjugated with monoclonal anti. Mycoplasma pneumonia . Proximity optical fibers were located around the viewing cell of the device, which were used to measure the increase in 45${\b{o}}$ forward light scattering of the immunoagglutinated particles. The detection limit was less than 50 $pgml^{-1}$ both for 390nm and 500nm microspheres with the detection time less than 90 seconds.

• 한국임상수의학회지
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• 제36권4호
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• pp.185-189
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• 2019

Respiratory pathogens of calves including bovine parainfluenza type 3 virus (BPI3V), bovine respiratory syncytial virus (BRSV), infectious bovine rhinotracheitis virus (IBRV) and Mycoplasma spp is well-known for winter pathogens. However, there are no studies about summer pneumonia pathogens of calves in Korea. The aim of this study was to detect respiratory pathogens from calves with summer pneumonia. Eighty calves from 5 regions were chosen and their nasal swabs were used to detect respiratory pathogens with real-time PCR. Mycoplasma spp was major primary respiratory pathogens in calves with summer pneumonia. Although, the detection rates of respiratory viruses were very low, serological assays showed that respiratory viruses exist widely in farms.

• 한국동물위생학회지
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• 제38권1호
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• pp.31-36
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• 2015

Two cats were obtained from a cat kennel. Over the previous 7 days, the cats had shown cough, anorexia, depression and nasal discharge. In this study, the consensus PCR was able to detect successfully Mycoplasma species in nasal swab samples of the cats. To identify feline mycoplasma species from the lung tissue of the cats with pneumonia, Mycoplasma species-specific PCR reactions were conducted. As the results, we could identify M. felis by the positive amplified DNAs. On the other hand, we could not detect any positive reactions with the PCR reaction for M. arginini, M. canis, M. edwardii, M. cynos, M. gateae, M. maculosum, M. molared, M. opalescens, M. spumans and Mycoplasma HRC-689. In conclusion, we detected M. felis from the kenneled cats with pneumonia. We suggested that this consensus PCR would be useful and effective for monitoring Mycoplasma species in various kinds of animals including cats. The application of preceding consensus PCR before the species-specific PCRs may be the most recommended strategy for the identification of Mycoplasma spp.

• 생명과학회지
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• 제17권2호통권82호
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• pp.279-285
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• 2007

위암 환자 30명과 결장암 환자 30명의 암 조직과 그 주위의 정상 조직을 구분하고, 암 조직과 정상조직의 쌍을 찾지 못한 경우의 위암 조직 8개와 결장암 조직 10개의 각 조직에서 Mycoplasma DNA의 존재 유무를 PCR법으로 확인하고 PCR산물에서 DNA의 염기서열을 분석하여 Mycoplasma DNA 서열과 비교함으로써 Mycoplasmas를 검색한 결과 다음과 같은 결론을 얻었다. 위암 조직과 그 주위 조직 30개 중에서 Mycoplasma DNA가 검출된 것은 각각 13개(43.3%)와 18개(60%)이었으며, 결장암 조직과 그 주위 조직 30개 중에서 Mycoplasma DNA가 검출된 것은 각각 12개(40%)와 15개(50%)이었다. Mycoplasma DNA가 검출된 위암 조직에서 mycoplasma 균종의 분리빈도는 M. faucium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae, M. conjunctivae의 순이었다. Mycoplasma DNA가 검출된 위암 조직 주변의 정상조직에서 mycoplasma 균종은 M. facium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae, M. bovigenitalium와 M. pulmonis의 순이었다. Mycoplasma DNA가 검출된 결장암 조직에서 mycoplasma 균종의 분리 빈도는 M. faucium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae), M. synoviae M. bovigenitalium, M. gallinarum, M. moatsii의 순이었다. Mycoplasma DNA가 검출된 결장암 조직 주변의 정상조직에서 mycoplasma 균종은 M. faucium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae, M. bovis, M. opalescens, M. bovigenitalium, M. gallinarum와 M. moatsii의 순이었다. 이상의 결과에서 위암 및 결장암 조직 보다 암 주위의 정상 조직에서 Mycoplasma DNA의 검출율이 높았으며, 검출된 Mycoplasma 균종도 암 조직과 정상 조직에서 차이가 없었으므로 이들 암과 Mycoplasma와는 상관관계가 없는 것으로 생각된다.

• Genomics & Informatics
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• 제3권3호
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• pp.63-67
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• 2005

Adaptive responses to diverse microbial pathogens might be limited in relatively few types. Host cell responses to pathogens are believed to be patterned or stereotyped along with species or class. We tried to compose the host response to Mycoplasma in terms of cellular gene expression. Although gene expression profile of two host HeLa and 293 cells were quite different each other, 30 genes were differentially expressed by mycoplasma infection in both of HeLa and 293 cells. Six of them (PR48, MADH4, MKPX, CRK, RBM7, NEK3) were related to cell cycle or proliferation. Another category of genes like IL1 HY1, KLRF1, TNFSF14, GBP1 were host defense to elicit immune responses. With this set of genes, we establish the prediction model for mycoplasma contamination.

• 한국임상수의학회지
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• 제35권6호
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• pp.273-275
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• 2018

A 6-month-old male cat was presented for investigation of depression, loss of appetite, dehydration, pale conjunctival mucous membrane, weight loss, fast heart and respiratory rates, nasal discharge and cough. Nasal swabs collected from the studied cat. As the results of bacterial culture with nasal swabs, it was suspected with Mycoplasma spp. Also, Mycoplasma species was detected by the PCR reaction with Mycoplasma genus primers. At species PCR assay, the specimens evaluated for the presence of M. felis, M. arginini, M. gateae, and Acholeplasma laidlawii and the result was visualization of bands from 238 bp in agarose gel 1.5% showing M. felis amplicons in samples. In conclusion, we detected M. felis in a cat with respiratory disease. PCR was able to detect successfully M. felis infection in cats.