title: 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.
authors.affiliation: International Patent Organism Depositary, AIST, Tsukuba, Ibaraki, Japan. yoko-ishikawa@aist.go.jp
authors.forename: Yoko
authors.initials: Y
authors.lastname: Ishikawa
authors.forename: Takaharu
authors.initials: T
authors.lastname: Kozakai
authors.forename: Hatsue
authors.initials: H
authors.lastname: Morita
authors.forename: Kaname
authors.initials: K
authors.lastname: Saida
authors.forename: Syuichi
authors.initials: S
authors.lastname: Oka
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Masuo
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D018929:Cell Culture Techniques; D004269:DNA, Bacterial / Q000032:analysis; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009174:Mycoplasma / Q000235:genetics*; D009175:Mycoplasma Infections; D009930:Organic Chemicals / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D051381:Rats; D012680:Sensitivity and Specificity; D013552:Swine
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I