• KSBB Journal
• /
• v.29 no.5
• /
• pp.361-371
• /
• 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.

• Korean Journal of Microbiology
• /
• v.41 no.4
• /
• pp.269-274
• /
• 2005

We evaluated the PCR assay and two commercialized PCR kits for the detection of mycoplasma in veterinary via live vaccines. The PCR assay could specifically detect all the tested Mycoplasma spp. and Acholeplasma spp., whereas two commercialized PCR kits did not. Also, the specificity of the PCR assay showed that 4 reference strains and 7 field isolates belonging to avian mycoplasma species could be all detected. The sensitivity of the PCR assay was determined using pure cultured Mycoplasma spp. and Acholeplasma spp. with a range of 1 to 100 colony forming units/ml in 9 CFR Mycoplasma broth. To test the availability of the PCR assay for veterinary live viral vaccines, A. laidlawii was artificially inoculated into the swine transmissible gastroenteritis-rota virus combined vaccine and canine parvovirus vaccine, respectively and the sensitivity of the PCR assay was similar with the result of cultured samples. In this study, the PCR assays could be used as rapid and sensitive methods for the detection of mycoplasma in veterinary live viral vaccines.

• Korean Journal of Veterinary Service
• /
• v.37 no.4
• /
• pp.247-252
• /
• 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.

• Korean Journal of Veterinary Research
• /
• v.39 no.1
• /
• pp.90-95
• /
• 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
• /
• 2009.07a
• /
• pp.187-199
• /
• 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.

• Journal of Veterinary Clinics
• /
• v.36 no.4
• /
• pp.185-189
• /
• 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.

• Korean Journal of Veterinary Service
• /
• v.38 no.1
• /
• pp.31-36
• /
• 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.

• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.279-285
• /
• 2007

Detection of Mycoplasma DNA from the 30 cases of cancer tissues and the normal tissues surrounding the cancer tissues obtained from the patients with gastric cancer and the other 30 cases of cancer tissues and the normal tissues surrounding the cancer tissues obtained from the patients with colon cancer were evaluated by polymerase chain reaction(PCR). The PCR products were sequenced using an ABI 377 automatic DNA sequencer, and these sequences were confirmed by comparing sequences with the database of the National Center for Biotechnology Information BLAST network server. Mycoplasmas DNA were defected in 18 (60%) cases of normal tissues which were around gastric cancer and were 13 (43.3%) cases of gastric cancer tissues. Mycoplasmas DNA were detected in 15(50%) cases of normal tissues which were around colon cancer and 12 (40%) cases of colon cancer tissues. The M. faucium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae, and M. conjunctivae were detected from the gastric cancer tissues. The M. faucium, M. subdolum,, M. salivarium, M. auris, M. hyosynoviae, M. bovigenitalium and M. pulmonis were detected from the normal tissues around gastric cancer. The M. faucium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae, M. synoviae M. bovigenitalium, M. gallinarum, and M. moatsii were detected from the colon cancer. The M. faucium, M. subdolum, M. salivarium, M. auris, M. hyosynoviae, M. bovis, M. opalescens, M. bovigenitalium, M. gallinarum, and M. moatsii were detected from the normal tissues around the colon cancer. These results suggest that Mycoplasmas infection may not correlate with gastric cancer and colon cancer, because of the detection rate of Mycoplasmas DNA were not significantly differences between normal and cancer tissues from the patients.

• Genomics & Informatics
• /
• v.3 no.3
• /
• pp.63-67
• /
• 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.

• Journal of Veterinary Clinics
• /
• v.35 no.6
• /
• pp.273-275
• /
• 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.