Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes

Abstract

Mycoplasmas (a generic name for Mollicutes) are a predominant bacterial contaminant of cell culture and cell derived products including viruses. This prokaryote class is characterized by very small size and lack of a cell wall. Consequently, mycoplasmas escape ultrafiltration and visualization under routine microscopic examination, hence the ease with which cells in culture can be contaminated, with routinely more than 10% of cell lines being contaminated. Mycoplasma are a formidable threat both in fundamental research by perverting a whole range of cell properties and functions and in the pharmacological use of cells and cell derived products. Although many methods have been developed, there is still a need for a sensitive, universal assay. Here is reported the development and validation of a quantitative polymerase chain reaction (qPCR) based on the amplification of a 1.5 kb fragment covering the 16S rDNA of the Mollicute class by real-time PCR using universal U1 and U8 degenerate primers. The method includes the addition of a DNA loading probe to each sample to monitor DNA extraction and the absence of PCR inhibitors in the extracted DNA, a positive mycoplasma 16S rDNA traceable reference sample to exclude any accidental contamination of an unknown sample with this reference DNA, an analysis procedure based on the examination of the melting curve and the size of the PCR amplicon, followed by quantification of the number of 16S rDNA copies (with a lower limit of 19 copies) when relevant, and, if useful, the identification of the contaminating prokaryote by sequencing. The method was validated on a collection of mycoplasma strains and by testing over 100 samples of unknown contamination status including stocks of viruses requiring biosafety level 2, 3 or 4 containments. When compared to four established methods, the m16S_qPCR technique exhibits the highest sensitivity in detecting mycoplasma contamination.

A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

“…In the case of the low level of infection, it is very difficult to identify correctly mycoplasma positive or negative samples [5,8,25]. Similarly, Jean et al showed that staining with the other DNA dye, Hoechst, only had a 25% sensitivity compared to the 95.2% sensitivity of qPCR [26]. Our results also clearly showed that even a relatively high concentration of mycoplasmas requires highly sensitive cameras for the valuable signal.…”

Characteristics of a novel cell line ZJU-0430 established from human gallbladder carcinoma

“…Mycoplasma can impede a whole range of cellular properties and functions and hinders the use of cells and their derived products in pharmacological area [46]. The culture supernatant derived from the ZJU-0430 cells was subjected to PCR analysis, and was found to be mycoplasma free.…”

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set

“…The strategy of designing primers at conversed sequences for selected livestock species allowed the development of a MSPS for potential use in the RT-qPCR assay. Although “universal” primers have been described in different contexts [30–33], to the best of our knowledge, a MSPS remained to be described for RT-qPCR applicable to multiple order-specific species and for inter-genus gene expression analysis. There are several evidences that the here described MSPS is useful for the livestock species; firstly, all ten primers detected as single amplicons by conventional RT-PCR in four different genera.…”

“…With the current relatively ease to generate large-scale gene expression datasets and the increasing demand for high-throughput or automated platforms [6,28,29], the development of tools for various species or technologies can become extremely advantageous. The use of a universal or multi-species primer set for PCR is an attractive approach to shortcut efforts to species-specific primer designing and testing [30–33]. To the best of our knowledge, despite the availability of a report for Capra hircus and Bos taurus [13], there is no multi-species primer set (MSPS) validated for intra-specific relative gene expression for several livestock species or for inter-genus RT-qPCR analysis.…”