Rapid and sensitive detection of Mollicutes in cell culture by polymerase chain reaction.
Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.
Shahhosseiny
Mohammad Hassan
MH
Department of Microbiology, Islamic Azad University, Shahryar-Shahr-e-Qods-Branch, Iran.
Hosseiny
Zahra
Z
Khoramkhorshid
Hamid Reza
HR
Azari
Shahram
S
Shokrgozar
Mohammad Ali
MA
D000127:Acholeplasma / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D018929:Cell Culture Techniques; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014509:Ureaplasma / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S