Scientific Publications by FDA Staff

Appl Microbiol Biotechnol 2007 Nov;77(1):223-32

Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT).

Kong H, Volokhov DV, George J, Ikonomi P, Chandler D, Anderson C, Chizhikov V

Volokhov DV (reprint author), US FDA, Ctr Biol Evaluat & Res, 1401 Rockville Pike,HFM-470, Rockville, MD 20852 USA US FDA, Ctr Biol Evaluat & Res, Rockville, MD 20852 USA American Type Culture Collect, Manassas, VA 20108 USA

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.