D. V. Volokhov, J. George, A. A. Neverov, H. Kong, D. Chandler, C. Anderson, V. E. Chizhikov, FDA, Rockville, MD
BACKGROUND: Mollicutes, which include the genera Mycoplasma and Acholeplasma, contamination is a common problem in research and production cell lines. Traditional systems of detecting these contaminants are resource intensive and time consuming. The use of rapid detection technologies would provide a major advantage in maintaining the purity and safety of biological products. This study describes development and evaluation of a PCR/Microarray that enables rapid and sensitive mycoplasma detection and accurate species identification of the contaminant.
METHODS: The current version of the microarray relies on the use of short oligonucleotide species-specific probes (20-30 nts in length) for identification of 25 species of Mycoplasma and Acholeplasma known to be human pathogens and common cell substrate contaminants. The assay procedure includes PCR amplification of the ITS and subsequent analysis of the amplified ssDNA or RNA using hybridization with the microarray. Two protocols, fluorescent dye and the nanogold particle staining, were developed for the preparation of the hybridization targets.
RESULTS: Broadly specific primers targeting the ITS were developed and efficiently used for amplifying more than 300 strains of Acholeplasma, Entomoplasma, Mycoplasma, Mesoplasma, Spiroplasma, and Ureaplasma species. Sequence analysis of these species revealed the presence of unique signature sequences in the ITS suitable for Mollicutes species identification using microarray technology and DNA sequencing. The use of nested PCR allowed us to increase the limit of detection to a few genomic copies of Mollicutes DNA per reaction. We demonstrated that our developed microarray was able to efficiently and unambiguously identify the species of target Mollicutes. We demonstrated that combining PCR/microarray with an initial step of in vitro enrichment, which includes 3-7 days cultivation of sample with Vero cells, we could significantly shorten the time and sensitivity of mycoplasma detection.
CONCLUSIONS: Sequence data showes that the 16S-23S rRNA ITS region is a suitable marker for Mollicutes detection and identification. Our designed PCR primers were very sensitive and highly specific. Evaluation of the microarray chips by hybridization with their appropriate target samples has demonstrated the feasibility of the PCR/Microarray method. The results demonstrated the potential of this highly sensitive and specific assay for detection and speciation of Mollicutes contamination in biological products and research applications.