Detection of multiple mycoplasma infection in cell cultures by PCR

Timenetsky, Jorge; Santos, Leandro Machado dos; Buzinhani, Melissa; Mettifogo, Elena

doi:10.1590/s0100-879x2006000700009

Cited by 74 publications

(64 citation statements)

References 29 publications

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“…Previously published endpoint PCR assays were developed for the detection of M. hyorhinis in cell culture, 30 in lung samples, 14,26 or in formalin-fixed, paraffin-embedded tissues from pigs with polyserositis. 12 In a 2014 published study, a qPCR was developed with a reported detection limit of 125 geq/μl.…”

Section: Discussionmentioning

confidence: 99%

Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

et al. 2014

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Abstract. Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

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Section: Discussionmentioning

confidence: 99%

Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

et al. 2014

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“…All strains biochemically and antigenically classified as Mycoplasma bovis were confirmed by the amplification of a 734 bp amplicon. Two oligonucleotide primers targeting the 16S rRNA gene, that are specific for M. bovis species were used: forward primer MboF2 (5'-GAAGAAAAAGTAGCATAGGAAATGAT-3') and reverse primer MboR2 (5'-CGTCGTCCC-CACCTTCCTCCCG-3') (Timenetsky et al 2006).…”

Section: Mycoplasma Bovis Isolationmentioning

confidence: 99%

Susceptibility of Mycoplasma bovis field isolates to antimicrobial agents

Šiugždaitė¹,

Gabinaitienė²,

Kerzienė³

2012

Vet. Med. - Czech

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ABSTRACT:The purpose of this study was to determine the antibacterial susceptibility of field isolates of Mycoplasma bovis originating from the upper respiratory tract of cattle of different ages. Bacteriological examination of 90 nasal swabs collected from calves at three months of age identified M. bovis in 31 (34.44%) samples. Seventeen (18.88%) of these animals still housed M. bovis in their nasal cavity at nine months and five animals (5.55%) still at seventeen months of age. M. bovis were confirmed by biochemical and antigenic methods. To confirm that these belonged to the M. bovis species isolated mycoplasmas were tested using the PCR method. Fifteen field strains of M. bovis isolated from the same cattle at three, nine and seventeen months (five strains from each age group) were selected for antibacterial susceptibility testing against six groups of antimicrobial agents using an agar dilution method. The MIC 90 ranges established for tylosin, tulathromycin, enrofloxacin, florfenicol and lincomycin were 0.39-0.78 µg/ml, 0.50-1.00 µg/ml, 0.78-1.56 µg/ml, 3.12 µg/ml and 0.39-0.78 µg/ml, respectively. The range of MIC 90 for oxytetracycline was from 50 to 100 µg/ml. Preliminary examination of the antimicrobial susceptibility of field strains of M. bovis did not reveal significant differences between different age groups of cattle. After evaluation of the MIC 90 data with the SPSS 13.0 statistical package it was found that M. bovis isolates from animals at three, nine and seventeen months were similarly susceptible to tylosin and tulathromycin. Statistically significant differences in susceptibility of M. bovis isolated from cattle of different ages were found to florfenicol compared with tulathromycin (P < 0.01), lincomycin (P < 0.01) and enrofloxacin (P < 0.05). The susceptibility of all M. bovis isolates to oxytetracycline and penicillin G significantly differed from the sensitivity to all other antimicrobial agents used in the present study (P < 0.05). The in vitro susceptibility test showed that field isolates of M. bovis isolated from cattle of different ages were similarly sensitive to tylosin, tulathromycin, lincomycin and enrofloxacin. It was also determined that the field strains are resistant to oxytetracycline.

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“…From more than 200 mycoplasma species, 20 have been isolated from cell cultures (Uphoff and Drexler 2013). Eight species including M. arginini, M. fermentans, M. hominis, M. orale, M. hyorhinis, M. salivarium, M. pirum and A. laidlawii are responsible for more than 95 % of cell culture contaminations (Harlin and Gajewski 2008;Markoullis et al 2009;Timenetsky et al 2006). The main sources of mycoplasma contamination in cell cultures are laboratory staff, serum, culture media, materials, reagents, instruments and previously contaminated cells.…”

Section: Introductionmentioning

confidence: 99%

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

et al. 2014

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Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert Ò and molecular. Enzymatic evaluation was performed using the mycoalert Ò kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert Ò method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (\20 min).

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Detection of multiple mycoplasma infection in cell cultures by PCR

Cited by 74 publications

References 29 publications

Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

Susceptibility of Mycoplasma bovis field isolates to antimicrobial agents

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

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