Table 1.
Indicator cell lines for mycoplasma detection using indirect Hoechst’ staining.
Table 2.
Sequences and optimal amounts of GFP and mycoplasma 16S primers for their use in real-time PCR.
Table 3.
Master mix GFP qPCR components.
Table 4.
Master mix m16S qPCR components.
Table 5.
Run program steps for the detection of 16S rDNA of mollicutes adapted from [5].
Fig 1.
Melting curves and amplification plots of 16S rDNA amplicons resulting from PCR using U1/U8 primers.
Melting curves obtained using p_m16S(0.9kb), a plasmid containing internally deleted 16S rDNA from M. capricolum subsp. capricolum strain California Kid (gi_83283139) (a), their reproducibility over multiple quantifications (b), with the amplification plot (c) and the linear regression analysis of Cq as a function of DNA copy input number (d, efficacy Em16S = 1.99847). For (c) and (d), 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 2 and 5 with DNA concentration measured by NanoDrop™ (http://www.nanodrop.com/).
Table 6.
Mean and SD values of CT and Tm from amplification plot and melting curves observed with p_GFP and p_16S(0.9kbp) used as DNA load and 16S positive controls respectively.
Fig 2.
m16S Melting curve (left, curves) and Cq (right, ladder) values obtained with a DNA panel from several mycoplasma species showing diversity of the melting curve from similarity with p_m16S(0.9kb) positive reference (a, b), different Tm (c) and bimodal curves (d).
(b) 1 M. imitans; 2 M. canis; 3 M.arginini, M. salivarium, M. alkalescens, M. agalactiae, M. canadense, M. apricolum subsps. capricolum; 4 M. mycoides subsp. capri; 5 M. putrefaciens; 6 M. verecundum; 7 M. moatsii, 8 M. fastidiosum; 9 M. alvi. (c) 10 M. ovipneumoniae; 11 M. iguana; 12 M. mycoides subsp. mycoides; 13 M. opalescens. (d) 14 M. yeatsii; 15 M. columborale; 16 M. lipofaciens, 17 M. fermentans. Tm peak of 16S rDNA amplicon is indicated by the dotted line on each graph.
Table 7.
Comparison of the sensitivity of five mycoplasma detection assays.
MeWo cells were inoculated with either culture medium (Medium) or 1.5 or 15 CFU of Acholeplasma laidlawii (A.l.). Cell free supernatants were collected after 5, 8 or 12 days of culture and tested with the various assays (see also Fig 4 for details).
Fig 3.
Relative sensitivity of qPCR and PCR using parameters optimized for qPCR.
(a) PCR product imaging after electrophoresis on agarose gel and staining. (b) Melting curves of qPCR samples (c) Table summarising data illustrated in (a) and (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.
Fig 4.
Comparison of the sensitivity of PCR and qPCR assays.
MeWo cells (1.5 x 106) seeded one day before in 25 cm2 tissue culture flasks were inoculated with either culture medium (Medium) or 1.5 or 15 CFU of Acholeplasma laidlawii (A.l.). Cell free supernatants were collected after 5, 8 or 12 days of culture (a) and DNA was extracted and analysed by qPCR and visualization of PCR products after electrophoresis on agarose gel (b-d). (see also Table 7).
Fig 5.
Melting curves and amplification plots of GFP DNA amplicons resulting from PCR using GFP.
Melting curves obtained using p_GFP (a), their reproducibility over multiple quantification runs (b) with the amplification plot (c), and the linear regression analysis of Cq as a function of DNA copy input number (d, efficacy EGFP = 1.99136) Tm peak of GFP amplicon is indicated by the dotted line. For (c) and (d), dilutions were done from a freshly prepared 99 bp PCR amplicon obtained from 5 pg of p_GFP using running conditions depicted in Table 2 with DNA concentration measured by NanoDrop™ (http://www.nanodrop.com/).
Fig 6.
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).
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 (a). Tm peak of 16S rDNA amplicon is indicated by the dotted line on each graph.
Fig 7.
Improvement of mycoplasma DNA detection by slowing the temperature ramping during amplicon melting.
Melting curve of sample #180 obtained after 100% ramping (a) and 10% ramping (b) with amplicon size determination (a,b, inset). Water and p_m16S(0.9kb) are shown in black and red dotted lines, respectively. Tm peak of 16S rDNA amplicon is indicated by the dotted line on each graph.
Fig 8.
Sensitivity and specificity of five mycoplasma detection methods.
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.
Fig 9.
Workflow diagram of decision making.