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.

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

Genotyping Measles Virus by Real-Time Amplification Refractory Mutation System PCR Represents a Rapid Approach for Measles Outbreak Investigations

Real-time PCR has been developed to genotype measles virus (MV) isolates. MV strains circulating in epidemics in Gabon in 1984, Cameroon in 2001, Morocco in 2003, and France in 2004 were investigated. We developed a real-time amplification refractory mutation system PCR (RT-AMRS PCR) using SYBR green fluorescent dye. Six pairs of primers for RT-ARMS PCR were designed to specifically amplify genotypes A, B2, B3.1, B3.2, C2, and D7. Genotypes could be differentiated by melting curve analysis. All strains were also confirmed by direct sequencing. Using the result obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of MV by RT-ARMS PCR and melting curve analysis was 97%. However, the latter method is more rapid and sensitive than the former method. This method could be a useful tool for molecular epidemiological studies of MV, providing an efficient alternative for large-scale studies

Melting curves and amplification plots of 16S rDNA amplicons resulting from PCR using U1/U8 primers.

<p>Melting curves obtained using p_m16S(0.9kb), a plasmid containing internally deleted 16S rDNA from <i>M</i>. <i>capricolum</i> subsp. <i>capricolum</i> strain California Kid (gi_83283139) (<b>a</b>), their reproducibility over multiple quantifications (<b>b</b>), with the amplification plot (<b>c</b>) and the linear regression analysis of Cq as a function of DNA copy input number (<b>d</b>, efficacy E_m16S = 1.99847). For (<b>c</b>) and (<b>d</b>), dilutions were done from a freshly prepared 0.9 kb PCR amplicon obtained from 5 pg of p_m16S(0.9kb) using running conditions depicted in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172358#pone.0172358.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172358#pone.0172358.t005" target="_blank">5</a> with DNA concentration measured by NanoDrop^™ (<a href="http://www.nanodrop.com/" target="_blank">http://www.nanodrop.com/</a>).</p

Relative sensitivity of qPCR and PCR using parameters optimized for qPCR.

<p><b>(a)</b> PCR product imaging after electrophoresis on agarose gel and staining. <b>(b)</b> Melting curves of qPCR samples <b>(c)</b> Table summarising data illustrated in (<b>a</b>) and (<b>b</b>). A cell culture supernatant known to be contaminated by mycoplasma was serially diluted and a sample of each dilution was run on qPCR. At the end of the qPCR run, the obtained qPCR product from each sample was analysed on agarose gel as standard PCR products.</p

Sensitivity and specificity of five mycoplasma detection methods.

<p>The sensitivity of the qPCR test was significantly better than the four other tests (***, p<6.5 x 10⁻¹² and below, Fischer’s test.). The specificity levels of all tests did not statistically differ (n.s., p>0.24 and above, Fischer’s test). See also material and methods section for details.</p

Run program steps for the detection of 16S rDNA of mollicutes adapted from [5].

<p>Run program steps for the detection of 16S rDNA of mollicutes adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172358#pone.0172358.ref005" target="_blank">5</a>].</p

Melting curves and amplification plots of GFP DNA amplicons resulting from PCR using GFP.

<p>Melting curves obtained using p_GFP (<b>a</b>), their reproducibility over multiple quantification runs (<b>b</b>) with the amplification plot (<b>c</b>), and the linear regression analysis of Cq as a function of DNA copy input number (<b>d</b>, efficacy E_GFP = 1.99136) Tm peak of GFP amplicon is indicated by the dotted line. For (<b>c</b>) and (<b>d</b>), dilutions were done from a freshly prepared 99 bp PCR amplicon obtained from 5 pg of p_GFP using running conditions depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172358#pone.0172358.t002" target="_blank">Table 2</a> with DNA concentration measured by NanoDrop^™ (<a href="http://www.nanodrop.com/" target="_blank">http://www.nanodrop.com/</a>).</p

Dissociation curves of m16S qPCR and amplicon size (insets) of DNA samples from cell-free supernatants and/or virus stocks and comparison with DNA from mycoplasma cultures when available with controls (a), samples (# follow by number) without detectable mycoplasma 16S rDNA (b) M. fermentans, (c), sample(s) with contaminated M. yeatsii (d) M. hyorhinis (e), A. laidlawii, (f), M. cottewii (g) and M. arginini (h).

<p>For sake of clarity, only corresponding amplicons run on agarose gel electrophoresis are shown in the inset with the 1 kb ladder markers (11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.65, 1, 0.85, 0.65, 0.6, 0.4, 0.3, 0.2 and 0.1 kb dsDNA) shown lane M in (<b>a</b>). Tm peak of 16S rDNA amplicon is indicated by the dotted line on each graph.</p

Comparison of the sensitivity of five mycoplasma detection assays.

<p>MeWo cells were inoculated with either culture medium (Medium) or 1.5 or 15 CFU of <i>Acholeplasma laidlawii (A</i>.<i>l</i>.<i>)</i>. Cell free supernatants were collected after 5, 8 or 12 days of culture and tested with the various assays (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172358#pone.0172358.g004" target="_blank">Fig 4</a> for details).</p