title: Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT).
abstract: Herein, we present data demonstrating that the application of initial cell culture enrichment could significantly improve mycoplasma testing methods based on the nucleic acid amplification technology (NAT) including a polymerase chain reaction (PCR)/microarray method. The results of the study using Vero cells demonstrated that this cell culture is able (1) to support efficient growth of mycoplasmas of primary interest, i.e., species found to be cell line contaminants, (2) to increase the sensitivity of NAT assay to the detection limits of the conventional broth/agar culture methods, and (3) to reduce the time required for mycoplasma testing fourfold in comparison with the conventional methods. Detection and identification of mycoplasmal agents were conducted using a modified PCR/microarray assay based on genetic differences among Mollicutes in the 16S-23S rRNA intergenic transcribed spacer (ITS). The application of nano-gold/silver enhancement technology instead of previously used fluorescent dyes significantly simplified the readout of microarray results and allowed us to avoid using expensive scanning equipment. This modification has the potential to expand the implementation of microarray techniques into laboratories involved in diagnostic testing of mycoplasma contamination in cell substrates and potentially in other biological and pharmaceutical products.
authors: Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, HFM-470, Rockville, MD 20852, USA.
authors: Hyesuk
authors: H
authors: Kong
authors: Dmitriy V
authors: DV
authors: Volokhov
authors: Joseph
authors: J
authors: George
authors: Pranvera
authors: P
authors: Ikonomi
authors: Donna
authors: D
authors: Chandler
authors: Christine
authors: C
authors: Anderson
authors: Vladimir
authors: V
authors: Chizhikov
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D002522:Chlorocebus aethiops; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D009174:Mycoplasma / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D036103:Nanotechnology; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D014709:Vero Cells
chemical_list: D021903:DNA, Ribosomal Spacer