There are numerous PCR-based methods for detection of Mycoplasma cited in the scientific literature. There are also a number of commercially available PCR-based assays that have the convenience of providing most of the reagents and controls needed to perform the assay and which appear to have high sensitivity for detecting Mycoplasma contamination. WHO doesn't endorse the use of any particular commercial assay, but the VenorGeM Mycoplasma Detection Kit is an example of a commercial kit which some laboratories find easy to use. VenorGeM is reported by the manufacturer to give a positive reaction with as little as 1–5 fg of Mycoplasma DNA (equivalent to 2–5 Mycoplasma organisms per sample volume). The VenorGeM kit is also reported to have ability to detect a range of Mycoplasma, including Acholeplasma and Ureaplasma species, both commonly found in contaminated cell cultures.
Have available the following items:
· 25 cm2 flask with cell culture to be evaluated for Mycoplasma;
· VenorGeM commercial kit containing:
- oligonucleotide primer set and nucleotides
- lyophilised primer set and deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP at optimized concentrations
- PCR 10X reaction buffer, sterile
- 100 mM tris-HCL (pH 8.5)
- 500 mM KCL
- 30 mM MgCl2
- Positive control DNA
- lyophilised DNA-fragment of Mycoplasma orale (non-infectious)
- Internal Control DNA
- lyophilised plasmid including Mycoplasma specific primer sequences and an internal sequence of the HTLV-1 tax gen with a size of approximately 191 bp
· Other materials/equipment needed but not provided in the VenorGeM kit:
- RNase-DNase free water
- 1U Taq DNA polymerase
- biosafety cabinet, BSC
- variable volume pipettors; 0.5-10 μl, 10-200 μl
- sterile ART tips
- thermocycler machine
- heating block
- screw-capped or locking tubes 1.5 ml (sterile)
- tube racks
- microcentrifuge
- microcentrifuge tubes
Test procedure
Preparation of samples
1) Working in a BSC, transfer 100 μl of medium from the 25 cm2 cell culture flask into a labelled, sterile 1.5 ml microcentrifuge tube. Discard the remainder of the medium from the flask.
2) Add approximately 0.5 ml of PBS to the flask and scrape cells from the flask into the PBS by using a cell scraper or an adapted sterile Pasteur pipette (create L-shape bend by softening with a bunsen burner). Mix the PBS and scraped cells to form a cell suspension.
3) Transfer 100 μl of cell suspension into a labelled, sterile, 1.5 ml microcentrifuge tube.
4) Incubate both samples (i.e. separate microcentrifuge tubes with cell suspension and medium) in a heating block at 95°C for five minutes.
5) Centrifuge (five seconds) the samples to pellet cellular debris and store at 4°C until ready for use.
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Good laboratory practice when doing PCR: Because the PCR technique involves amplification, PCR-product carryover (cross-contamination) represents a significant problem. Observing the following good laboratory practices can diminish the problem: · Use a separate room or containment unit (biological safety cabinet equipped with UV light) for pre- and post-PCR procedures. · Use separate sets of pipettors and other equipment for pre-and post-PCR procedures. · Aliquot reagents and store to minimize the number of repeated samplings. · Prepare and aliquot reagents in an area that is free of PCR amplified products. · Always use aerosol-resistant tips. · Wear gloves (talc free) and change frequently. · Uncap tubes carefully to prevent aerosols. · Minimize sample handling. · Add non-sample components to the reaction tubes before adding the sample and controls. · Cap each tube after the addition of sample before proceeding to the next sample. · Use a positive control that amplifies consistently. · Use a negative control. · Include one or more reagent controls with each amplification. Reagent controls should contain all of the necessary components for PCR except the template RNA. |
PCR reactions
1) Rehydrate PCR primer set, nucleotides and positive control DNA in a PCR-Clean Area.
- Before rehydration, briefly centrifuge all tubes to ensure that lyophilised pellets are spun down.
- Use the following procedure when rehydrating materials: add the appropriate amount of H2O and allow the tube to sit at room temperature for 5 minutes. Mix the solution by vortexing a few seconds or by pipetting up and down repeatedly to completely dissolve the DNA. Briefly centrifuge again.
- Rehydrate PCR primer set and nucleotides with 130 μl sterile RNase-DNase free H2O. Aliquots of primers and nucleotides should be prepared and stored frozen until ready for use.
- Rehydrate positive control with 100 μl sterile RNase-DNase free H2O.
- Rehydrate internal control with 220 μl of sterile RNase-DNase free water.
2) Prepare the PCR master mix in sterile 1.5 ml tubes, preparing sufficient volume to allow 48 μl per sample and control:
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Reagents Volume per reaction 10 x PCR reaction buffer 5.0 μl Mycoplasma Primer set and dNTPs 5.0 μl Taq DNA polymerase 0.2 μl Internal Control DNA 2.0 μl RNase-DNase free water 35.8 μl Total volume 48.0 μl |
Aliquot 48 μl of master mix into each PCR reaction tube required, including tubes for controls.
3) Add samples and controls to the PCR master mix in the sample preparation area:
- Have available the samples, controls and master mix on a rack in an ice bath.
- Working in a BSC, add 2 μl of each sample and control to appropriately labelled PCR tubes containing reaction mix. Include 2 μl of kit positive control and 2 μl of RNase-DNase-free water as a negative control.
4) Run samples on Thermocycler using the following parameters. Run time is approximately three hours:
- Cycle 1: 94°C for 2 minutes, 55°C for two minutes, 72°C for three minutes;
- Cycle 2 to 35: 94°C for 30 seconds, 55°C for one minute, 72°C for one minute;
- hold at 72°C for four minutes;
- hold at 4°C until tubes are removed.
5) Run 10 μl of all PCR amplified samples on 2% agarose gel at 110V for one hour. Include a 100 base pair ladder.
6) Stain gel with ethidium bromide and photograph for permanent record.
Interpretation
The results are interpreted by comparing the presence and size of PCR products from test samples to those of positive control reactions. The positive control should show a strong 270-bp band and the negative control should show no bands in this region. The Internal Control DNA should produce a 191 base pair band, indicating no inhibition of the PCR reaction. Any samples containing Mycoplasma DNA will produce a distinct and usually strong 270 base pair band, i.e. at the same position as seen with the positive control. Uninfected samples will show no band at this size. The VenorGeM Mycoplasma assay is designed for high sensitivity and is therefore prone to non-specific annealing. Bands of various lengths that are less intensive can be produced but do not indicate positive results. Self-annealing of primers can produce a produce of 80–99 base pairs, but this also does not affect the precision or results of the test.