Abstract:Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine… Show more
“…The use of two methods has been the most recommended strategy to minimize false results. PCR in combination with culture is the most widely recommended procedure (30). In the present study, PCR detected more infected samples than culture.…”
A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.
“…The use of two methods has been the most recommended strategy to minimize false results. PCR in combination with culture is the most widely recommended procedure (30). In the present study, PCR detected more infected samples than culture.…”
A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.
“…The presence of false positive results is considered as an important disadvantage of PCRbased methods especially in nested PCR (Cheong et al 2011). Although the hybridization technique is basically established on PCR method principles, (Uphoff and Drexler 2002). In a previous study by Mc Garrity et al mycoplasma contamination was evaluated in 30 cell lines using microbial and PCR methods, which respectively detected 10 and 14 positive ones.…”
Section: Discussionmentioning
confidence: 99%
“…The LOD of nested PCR was similar to the sensitivity of microbial culture and higher than for Mycoalert Ò . Uphoff and Drexler also reported that in comparison with microbial culture and DNA-RNA hybridization, molecular PCR technique could be considered as a sensitive, economic, accurate, precise, cheap, simple and rapid method for detection of mycoplasma contamination in cell cultures (Uphoff and Drexler 2002). On the other hand, according to the Young et al suggestion, at least two or three tests must be synchronously performed for contamination recognition (Young et al 2010).…”
Section: Discussionmentioning
confidence: 99%
“…Data were analyzed by t test for comparing two tests and Freidman test for comparing three tests. Sensitivity, specificity, accuracy and predictive value of positive and negative results were calculated using Microbial Culture as the Gold Standard (Hopert et al 1993;Uphoff and Drexler 2002;Galen and Gambino 1975 …”
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).
“…1 Mycoplasma contamination has been detected in 15-35% of cell lines deposited in some cell culture collections. 2 Although the manufacture of recombinant proteins and vaccines from cell lines has a good safety record, there have been reports of the contamination of production processes and viral vaccines by viruses most probably derived from contaminated media components and fetal calf sera. 3 -7 Porcine parvovirus has been isolated from commercial trypsin used in passaging of adherent cell lines.…”
Replication competent oncolytic viruses, like other biological products, are at risk from contamination by bacteria, fungi, mycoplasma and viruses that must be eliminated from the final product. This article reviews the regulatory guidance for the manufacture and testing for oncolytic virus products. A testing strategy covering the testing of cell lines, virus banks, virus harvests and purified product is described.
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