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.

Jeffrey Chalmers,Audrey Jean,Isabelle Grosjean,Denis Gerlier,Omran Allatif,Bariza Blanquier,Florence Tardy

doi:10.1371/journal.pone.0172358

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.

Highlights

Cells lines and primary cell cultures are very frequently used as tools to unravel the molecular and cellular mechanisms that underlie biological processes, such as cell-invasion by viruses, microbes or parasites
To validate the m16S_qPCR, hundreds of samples from either cell culture or BSL2 to BSL4 viral stocks were tested for mycoplasma contamination by this technique and whenever possible, compared with four other assays— Hoechst DNA staining, MycoAlert and PlasmoTest and polymerase chain reaction (PCR)
Because of the need of many colleagues working with cell lines and viruses requiring BSL2 to BSL4 biosafety levels, the aim was to find and to implement a mycoplasma detection assay sensitive enough that it could be used universally

Summary

Introduction

Cells lines and primary cell cultures are very frequently used as tools to unravel the molecular and cellular mechanisms that underlie biological processes, such as cell-invasion by viruses, microbes or parasites. To validate the m16S_qPCR, hundreds of samples from either cell culture or BSL2 to BSL4 viral stocks were tested for mycoplasma contamination by this technique and whenever possible, compared with four other assays— Hoechst DNA staining, MycoAlert and PlasmoTest and PCR.

Results

Conclusion