Rapid Detection of Mycoplasma Contamination in Cell Cultures Using Sybr Green-Based Real-Time Polymerase Chain Reaction

Abstract

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

“…Despite the reported ability of this mycoplasma testing procedure to efficiently detect all possible cell culture mycoplasmal contaminants, the overall testing procedure is time-consuming (a minimum of 28 days) and tedious and might not be suitable for biologics with shelf-lives that are shorter than the turnaround time for testing. In order to reduce the time required for mycoplasma testing, various approaches based on nucleic acid testing (NAT) technologies targeting different genetic markers of Mollicutes have been developed and proposed as potential alternatives to the current methods (15,31,33,54,56,57,63). However, although PCR methods have definite advantages over conventional microbiological methods in terms of analytical sensitivity, simplicity, and turnaround time required for testing, it continues to be unclear whether PCR-based methods can provide a limit of detection comparable or superior to those of conventional methods.…”

A single-tube real-time PCR assay for mycoplasma detection as a routine quality control of cell therapeutics

“…As previously shown [11][12][13][14], real-time PCR provides a highly sensitive and rapid technique for mycoplasma detection in cell culture or clinical samples. Based on the concept of a conventional PCR assay described in our previous study [ …”

“…More recently, a number of assays based on real-time PCR have been introduced [11][12][13][14]. Whereas Störmer et al [14] used conserved regions of the tuf gene for development of a broad-range PCR assay, most of the PCR assays target the 16S rDNA region.…”

A Single-Tube Real-Time PCR Assay for Mycoplasma Detection as a Routine Quality Control of Cell Therapeutics

“…As previously shown [11,12,13,14], real-time PCR provides a highly sensitive and rapid technique for mycoplasma detection in cell culture or clinical samples. Based on the concept of a conventional PCR assay described in our previous study [3], we now present a single-tube real-time PCR assay with less hands-on time suitable for routine testing.…”