High-throughput detection and multiplex identification of cell contaminations

Abstract

Unnoticed cell culture contamination by viruses, Mycoplasma, or other cell lines is not uncommon and a threat to laboratory safety and the quality of scientific results. We developed and validated a novel high-throughput Multiplex cell Contamination Test (McCT), which is currently able to detect 37 contamination markers in a single reaction. The assay is based on multiplex PCR with target-specific primers and subsequent hybridization of amplimers to specific oligonucleotide probes. McCT proved to be highly specific, sensitive and robust, and allows to analyze more than 1000 cell lysates per week. In conclusion, the novel McCT assay is a powerful high-throughput tool in assessing cell line purity.

Figure 1.

Schematic overview of the McCT (picture of the Luminex analyser taken from Luminex Corp. webpage).

Figure 2.

Detection of Mycoplasma (M.) dilutions by Venor®GeM (A) and McCT (B). Both assays were performed using 6-fold dilution series of one cell lysate positive for M. arginini and M. hyorhinis. (A) PCR products from the commercial Mycoplasma kit were loaded on a 2% agarose gel showing the internal PCR control (191 bp) and a Mycoplasma-specific band (267 bp). (B) Net MFI values obtained after hybridization of the McCT PCR products to Mycoplasma species-specific probes are shown. The cut-off is indicated by the dotted line.