The authors have declared that no competing interests exist.
Mycoplasmas (a generic name for
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. In order to avoid biased interpretation of cell-based experiments, these tools should be kept under stringent quality scrutiny. Together with errors in cell line authentication, contamination by mycoplasmas is recognized as one of the two major pitfalls in cell culture. To give just one example, a survey of transcriptomic data deposited in NCBI Sequence Read Archive has shown that more than ten percent of the samples contained identifiable RNA from mycoplasmas meaning that many transcriptomic data have been published though being invalid [
Detecting a mycoplasma contamination is not straightforward. Gold standards look for growth of mycoplasma colonies cultured on broth agar over several weeks and search for extra-nuclear DNA dots stained with Hoechst’s reagent. In both cases, this means days or weeks of culture to allow the growth of the mycoplasma until the colonies reach a size large enough to be seen macroscopically and microscopically, respectively. Other techniques have been developed, such as enzymatic- and bio-assays, ELISA, and polymerase chain reaction (PCR). These techniques are either cumbersome, difficult to interpret, of limited sensitivity and/or limited to the detection of only a limited range of species [
Supernatants of cell culture were prepared by harvesting cell free supernatants and further clearance of cell debris by centrifugation in 15 mL conical tubes at 200 g at room temperature for 5 minutes. They were kept frozen at -80°C until use. All samples were manipulated under a Type II laminar flow and biosecurity level laboratory containment (BSL2, 3 or 4) as required for the manipulation of cells and viruses until their full inactivation. Samples include routine checking for mycoplasma contamination in cell lines and virus stocks (or infected cells) of RNA viruses (measles, canine distemper, vesicular stomatitis, Ebola, Nipah, influenza, Crimean-Congo haemorrhagic fever, human T lymphotropic I, Drosophila C, Drosophila X, Mopeia, Puumala, Gypsy virus) and DNA viruses (Epstein Barr, BK virus).
The principle is to detect mycoplasma colonies growing adjacent to cells by visualizing DNA dots located outside the cell nuclei. The indirect assay was used as described previously [
Name | Species | Culture medium |
cells/well |
---|---|---|---|
MeWo [ |
Human | RPMI 1640 Glutamax-I, 10% fetal calf serum, 1% Sodium pyruvate, 1% non-essential amino acids, 10 mM HEPES pH7.2 | 4x104 |
Vero [ |
Monkey | DMEM GlutaMax-I,10% fetal calf serum | 8x103 |
IgH-2 [ |
Iguana | EMEM, 2 mM Glutamax, 10% fetal calf serum 1% non-essential amino acids | 6x103 |
NIH3T3 [ |
Mouse | RPMI 1640 Glutamax-I, 5% fetal calf serum | 4x103 |
CHO [ |
Hamster | Ham’s F-12 Nutrient Mixture, 10% fetal calf serum | 4x103 |
1 in humidified incubator, in the presence of 5% CO2 at 37°C
2 amount of cells to be seeded in 2 mL of culture medium in 6 well-plates.
The principle is to detect a mycoplasma specific enzyme using a luciferase-based assay. The procedure was performed as recommended by the manufacturer (
The principle is to detect mycoplasma lipopeptides by Toll like receptor 2 (TLR2). This requires the use of a HEK-Blue-2 reporter cell line that stably expresses TLR2, which upon binding to a lipopeptide agonist activates the secretion of alkaline phosphatase. The secreted enzyme is detected by colorimetry. The procedure was performed as recommended by the manufacturer (
The p_GFP plasmid coding for the green fluorescent protein described elsewhere [
The 1514 bp 16S rDNA fragment from
Primer name | Primer sequence |
Final concentration | Reference |
---|---|---|---|
16S U1 (forward) | 1 μM |
[ |
|
16S U8 (reverse) | 2 μM |
[ |
|
GFP_for | 0.2 μM | [ |
|
GFP_rev | 0.2 μM | [ |
*
** or 0.17 μM of each individual sequence assuming equal proportion of concatenated nucleotide species at the degenerate position during oligonucleotide synthesis
*** that is 0.125 μM of each individual sequence
Critical step: Use Low Binding filter tips and tubes to maximize sample recovery and avoid DNA cross-contamination.
Add 10 μL of p_GFP (10 pg/μL) as DNA loading tracer to 1 mL of cell-free supernatant sample into 1.5 mL Low Binding conical tube, and vortex.
Centrifuge at 8,000 g exactly for 5 min at RT.
Carefully discard the supernatant to spare the very small pellet (maybe difficult to see)
Lyse the pellet using 180 μL of Lysis Buffer T1 from the NucleoSpin Tissue kit (Macherey-Nagel, cat. No. 740952) by repeating pipetting.
Add 25 μl of Proteinase K and vortex vigorously.
Incubate at 56°C for at least one hour with frequent vortexing.
Add 200 μL of Lysis Buffer B3 and vortex vigorously.
Incubate at 96°C for 15 min.
Vortex briefly.
Add 210 μL of absolute ethanol and vortex vigorously. White filaments may appear.
Transfer the whole sample (~500 μL including white filaments) onto a NucleoSpin Tissue column above a collector tube.
Centrifuge at 11,000 g for 1 min at RT.
Discard the flow through solution from the collecting tube.
Add 500 μL of Wash Buffer B5 on the column.
Centrifuge at 11,000 g for 1 min at RT.
Discard the flow through solution from the collecting tube.
Add 600 μL of Wash Buffer B5 on the column.
Centrifuge at 11,000 g for 1 min at RT.
Discard the flow through solution from the collecting tube.
Centrifuge at 11,000 g for 1 min at RT and throw the collector tube.
Put the column on the top of a 1.5 mL Low Binding conical tube.
Add 50 μL of Elution Buffer BE heated to 70°C on the column and incubate for 3 min at RT.
Centrifuge at 11,000 g for 1 min at RT.
Add 50 μL of Elution Buffer BE heated to 70°C on the column and incubate for 3 min at RT.
Centrifuge at 11,000 g for 1 min at RT.
Aliquot the DNA solution in four 0.5 mL Low Binding conical tubes (25 μL/tube) and kept them frozen at -20°C until use.
Discard the flow through solution from collecting tube.
Carry out a master mix with primers "GFP" by adding reagents as shown in
Master Mix GFP | Final Concentration | Volume per reaction tube (μL) |
---|---|---|
4.6 | ||
1X | 10 | |
0.2 μM | 0.2 | |
0.2 μM | 0.2 |
(see
The qPCR is performed as described above for GFP DNA except for the composition of the SYBR Green/Primer mix.
Prepare the master mix for the primers "m16S" by addition of reagents as shown in
Master Mix m16S | Final Concentration | Volume per reaction tube (μL) |
---|---|---|
2 | ||
1X | 10 | |
1 μM | 1 | |
2 μM | 2 |
(see
Vortex, then shortly centrifuge the SYBR Green/Primer mix to eliminate any foam.
Add 15 μL of master mix “GFP” or “m16S to each well of a MicroAmp Fast optical 96-well Reaction Plate (Applied Biosystems, cat. no. 4346906).
Add in duplicate 5 μL of either water, or p_GFP (at 1 ng/mL) or p_m16S (at 1 ng/mL) or samples.
Carefully close each well by covering the plate with one MicroAmp Optical adhesive Film.
Centrifuge shortly the plate for 10 s to bring mixtures at the bottom of the well.
Run the qPCR according to
Cycle Number | Step | Temperature | Period | |
---|---|---|---|---|
Activation of the enzyme | 95°C | 10 min | ||
Denaturation | 95°C | 15 s | ||
Hybridation / Elongation | 65°C | 2 min | Data collection |
Data are collected and curves are generated with StepOne software (Applied). Each reaction is carried out in duplicate. The analysis of amplicon strand dissociation is performed at the end of the run to visualize amplification specificity.
To optimize the melting curve, the fluorescence is acquired at every 0.3°C during a 65°C to 95°C temperature gradient.
Quantification cycle (Cq) method determination: manually set the threshold above the background on the lower limit of exponential phase of kinetic amplification. Cq is the crossing point between threshold and kinetic.
In order to determine primers efficiencies, a template qPCR reaction is performed for each primer couple and then diluted to generate linear standard curves. Primer efficiencies are reported in
Melting curves obtained using p_m16S(0.9kb), a plasmid containing internally deleted 16S rDNA from
Relative quantities of DNA copies are calculated using primer efficiency (e) according to the mathematical model of Pfaffl [
In the case of doubtful results from the analysis of the melting curve, the qPCR programme is modified by adding a 10% ramping time between denaturation at 95°C and hybridization/elongation time at 65°C. At the end of the PCR, the plate is kept frozen until further use.
The amplicons generated during the m16S_qPCR were analysed by electrophoresis in an agarose gel essentially as detailed in [
Amplicons of 1.5 kb size were recovered using NucleoSpin Gel and PCR Clean-up kit (
They were calculated as reported [
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. Within CelluloNet BioBank, the indirect Hoechst staining assay based on the human MeWo cell line [
To establish a Real-Time PCR with U1/U8 degenerate primers, the 1.5 kb long 16S rDNA from
Primers | DNA source | Number of measurements | CT (mean ± SD) | Tm (mean ± SD |
---|---|---|---|---|
GFP | water | 17 | 27.7 ± 1.4 | 76.4 ± 0.26 |
p_GFP | 15 | 17.6 ± 1.5 | 83.5 ± 0.05 | |
16S U1/U8 | water | 21 | 34.4 ± 2 | 74 ± 0.44 |
C16S p_m16S(0.9kbp) | 18 | 19 ± 1.6 | 80.8 ± 0.15 |
The ability of m16S_qPCR to detect and to quantify available DNA stocks from an internal mycoplasma collection was then tested. All samples with measurable DNA contents using Qubit dsDNA HS Assay Kit (ThermoFischer Scientific, cat. No. Q32851), i.e. >0.4 ng/mL, gave a measurable signal. Amplicons from most mycoplasma strain samples gave a melting curve that was identical with that observed with p_m16S(0.9kb) positive control (
(
As the qPCR assay was using the same set of U1/U8 primers previously recommended for detection of mycoplasma [
MeWo cells were inoculated with either culture medium (Medium) or 1.5 or 15 CFU of
Seeding at day 0 | Days in culture | MycoAlert™ | PlasmoTest™ | Hoechst | PCR | qPCR | |
---|---|---|---|---|---|---|---|
Detection | Quantification | ||||||
Medium | 5 | N | N | N | N | N | NQ |
8 | N | N | N | N | N | NQ | |
5 | N | N | N | N | Y | NQ | |
8 | N | N | N | N | Y | NQ | |
12 | N | N | Y | N | Y | NQ | |
5 | N | N | Y | Trace? | Y | NQ | |
8 | N | N | Y | Trace? | Y | NQ | |
12 | N | N | Y | Y | Y | 1,29 x 105 |
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
To evaluate to what extent m16S_qPCR can be routinely used to detect mycoplasma contamination of cell cultures and virus stocks, about one hundred cell-free supernatants or virus stocks were analysed and the results were compared with those obtained using the indirect Hoechst staining, MycoAlert and/or PlasmoTest detection assays whenever technically possible. Since the first step of m16S_qPCR required DNA purification from the unknown samples, a small amount of p_GFP was added to each sample prior to DNA extraction. The use of this DNA loading probe allowed us to ensure recovery of DNA free from PCR inhibitors from every sample by running GFP-specific qPCR as illustrated in
Melting curves obtained using p_GFP (
DNA extracted from most of the unknown samples and run using m16S_qPCR showed only the melting curve of the primer dimer (
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 (
In 4 samples, the melting curve profile did show a small shoulder at a higher temperature than that of the melting curve of the primer dimer (
Melting curve of sample #180 obtained after 100% ramping (
A few other procaryotes can be also detected with the PCR as predicted by blasting U1 and U8 primers against bacterial genome databases (see
Among 87 cell-free supernatant or virus stock samples tested, ~17% were found positive for mycoplasma contamination by m16S_qPCR. A few samples (4.6%) could not be tested by any of the other three methods because of technical constraints. From the qPCR positive samples (that were also tested with one, two or three of the other assays, 4.6% were also detected as being contaminated by mycoplasma and 8.05% escaped detection by one or two of other assays. Furthermore, 8 samples in which no 16S rDNA could be detected by m16S qPCR were found to give positive signals by one assay (or even by 3 different assays for one sample) and 2 with doubtful results for at least one assay (11.5%). The 10 samples giving just above threshold signals by MycoAlert could not be confirmed using this assay upon testing of cells that have been infected by these viral stocks and it was speculated that concentrated stocks of enveloped viruses may contain a cell-derived enzymatic source of ATP resulting in possible signal bias using this test.
By taking into account the samples tested with their mycoplasma positive or negative status, the sensitivity and specificity were independently calculated for each of the five methods used to detect mycoplasma (
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.
In conclusion, the m16S_qPCR method to track contamination of cultured cells and cell derived products associates high sensitivity, a very broad range covering the entire
Step 1: A known amount of p_GFP as DNA loading probe is added to cell-free samples to be tested and DNA is purified.
Step 2: The efficiency of DNA purification and the absence of PCR inhibitors is determined by GFP-specific qPCR. In case of low or abnormal GFP signal, DNA purification is performed again.
Step 3: m16S-qPCR is run on the DNA sample, water as negative control and p_m16S(0.9kb) as reference positive DNA.
Step 4: Amplicon melting curve is analysed.
- 4(a) a melt curve identical to primer indicates lack of detectable mycoplasma contamination (<19 copies/sample) and no further analysis is required. - 4(b) an atypical melt curve with a visible shoulder peaking around 81°C that suggests a 16S rDNA signal, go to step 6.* - 4(c) a melt curve nearly identical to that obtained with p_m16S(0.9kb) positive control indicates mycoplasma contamination; go to step 5 for quantification.
Step 5: Cq plotting on standard curve gives the contamination level
Step 6: Check amplicon size by agarose gel electrophoresis
- 6(a) 1.5 kb amplicon size: mycoplasma (or bacteria) contamination is confirmed. Go to step 7. - 6(b) no signal corresponding to a 1.5 kb amplicon size with small Tm shoulder at ~81°C indicates a low mycoplasma contamination. - 6(c) 0.9 kb amplicon size: accidental contamination with 16S rDNA standard: go to Step 1 to run again the sample.
Step 7: Amplicon can be sequenced for identification of the prokaryote contamination.
* Note in case of a very low shoulder with a Tm ~81°C the m16S-qPCR can be run again on the sample but using the 10% ramping protocol (Step 8).
The universal m16S_qPCR procedure has several advantages over published available methods including those that are also based on PCR or multi-primer qPCR [
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This work was supported by a two-year grant from the Infrastructure BIOBANQUES (INSERM US013) and IBiSA (AAP2013) to CelluloNet BioBank, BB-0033-00072 (DG). The authors thank the following people for their commitment in providing virus stocks, cell supernatant or bacteria and planta samples and/or performing tests requiring BSL3 or BSL4 containment: C. PEREIRA, J. BRUNEL, C. MATHIEU (CIRI team “Immunobiology of Viral Infections”), Y. BENITO, L. BERAUD, (CIRI team “Staphylocococcal Pathogenesis”), O. REYNARD, (CIRI team “Molecular Basis of Viral Pathogenicity”), S. ALAIS, (CIRI team “Retroviral Oncogenesis”), H. GRUFFAT, (CIRI team “Oncogenic Herpesviruses”), D. DECIMO (CIRI team “Eukaryotic and Viral Translation”) J. HOFFMANN, M. MOROSO, M. MILENKOV, (CIRI team “Emerging Pathogens Laboratory”, Fondation Mérieux), L. LINES, I. PILA-CASTELLANOS (CIRI team “Cell Biology of Viral Infections”), S. REYNARD (CIRI team “Unit of Biology of Emerging Viral Infections”, Institut Pasteur), V. BARATEAU, (CIRI team “Host-Pathogen Interaction during Lentiviral Infection”), E. DECEMBRE (CIRI team “Vesicular trafficking, innate response and viruses”), F. FAURE (CIRI team « Innate Immunity in Infectious and Autoimmune Diseases »), F. GUIGUEN, Christophe TERZIAN (INRA, UMR 754, RPC team “Infection and Endogenous Retroviruses”), S. MELY, D PANNETIER (Laboratoire P4 Jean Mérieux, INSERM, US3), and Nicolas SAUVION (INRA BGPI, Montpellier). The authors also thank R. CATTANEO and P. DEVAUX for providing us with useful plasmid reagent, Xavier FOISSAC for helpful discussions, and acknowledge the support of the Measles virus BioBank BB-0033-00053 facility and the BSL3-ANIRA facility of the SFR BioSciences, INSERM, CNRS, UMS3444/US8. Philip Lawrence is also thanked for English language revision.