Comparative PCR Analysis for Detection of Mycoplasma Infections in Continuous Cell Lines

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 the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.

Detection of multiple mycoplasma infection in cell cultures by PCR

“…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.…”

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

“…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.…”

“…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).…”

“…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 …”

Biosafety and product release testing issues relevant to replication-competent oncolytic viruses

“…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.…”