Mycoplasma Test Kits
PCR kits for sensitive and rapid detection of mycoplasmas in cell culture
PCR Mycoplasma Test Kit I/C (for conventional PCR)
PCR Mycoplasma Test Kit I/RT (for Real-Time PCR)
PCR Mycoplama Test Kit II (for conventional PCR)
PCR Mycoplama Test Kit III (for conventional PCR)
Background
Several methods for the detection of mycoplasmas have been published:
- Cultures on agar, liquid media, or semi-solid media
- Staining with DAPI or other fluorescent dyes (e.g. 4', 6-diamine-2-phenylindole dihydrochloride)
- DNA hybridization
- Antibodies for specific mycoplasma species
- Electronic microscope
- PCR: specific primers
The testing required by the regulatory authorities is seeding in culture (agar and liquid media). This test is complicated, time consuming (about 5 weeks), and some mycoplasma species are difficult to detect with this method. In recent years, the disadvantages of these methods have been acknowledged (such as sensitivity, specificity and long and complex procedures), and use of PCR for the detection of contaminations in cell cultures has become increasingly widespread
Using PCR for the Detection of Mycoplasmas
PCR has been shown to be a highly sensitive, specific and rapid method for the detection of mycoplasmas contamination in cell cultures. Specific primers have been designed from DNA sequences that are coding for highly conserved ribosomal RNAs (16S-rRNA). The gene sequences for those conserved rRNAs are very similar in the various mycoplasma species and have not undergone significant mutations. Consequently, primers can be designed for these areas, which are specific to the contaminating mycoplasmas only and will not detect bacterial or animal DNA sequences.
The literature describes several PCR methods for the detection of mycoplasmas, such as using a number of primers to obtain detection of specific mycoplasma species or nested PCR (two consecutive PCR cycles using different primers) for amplifying sensitivity and specificity. PCR testing techniques are all based on amplification of a distinct DNA fragment using specific primers prepared in advance, and fragment identification is usually achieved by gel electrophoresis or real-time PCR.
PCR-based mycoplasma test kits greatly simplify testing and detection of mycoplasma contamination in cell cultures. With PCR testing, reliable results are obtained within a few hours, since the presence of contaminant mycoplasmas can be easily and sensitively detected by simply verifying the bands of amplified DNA fragments after gel electrophoresis.
Detection of Mycoplasmas: PCR Based Method versus Culture Method
PCR-BASED METHOD:
1. Rapid - results within 5 hours
2. Simple, one-step reaction - less than 10 minutes to prepare the sample. The mixture contains everything (including Taq Polymerase!)
3. Can be used routinely in every lab
4. Minimal sample handling reducing the risk of contamination
5. Highly sensitive (~10-1000 CFU/ml)*
6. M. hyorhinis can be detected
MICROBIOLOGICAL CULTURE:
1. Long and time consuming - results up to 3 weeks
2. Cumbersome
3. Requires experienced personnel
4. Sensitive
5. Hard to detect M. hyorhinis
* Adequate to diagnose cell cultures infected with mycoplasmas. Infections usually result in mycoplasma titers of 105 - 108 CFU/ml (McGarrity G. et al., 1982)
Screen frequently to validate your experimental results
Mycoplasma contamination is a recurring problem that can cause non-reproducible, questionable results in research and productivity losses of up to 50% in industry - but can be avoided by routine diagnostic testing. However, only one of ten scientists regularly check their cell cultures for mycoplasma contamination. Nevertheless, testing for mycoplasma infection is now an essential requirement in reaching quality control standards for laboratories concerned with the production and maintenance of cell lines used in manufacturing various biologicals. Screening of cell cultures at regular intervals for mycoplasma contamination is critical for reliable and reproducible results. We recommend to screen cell cultures on a monthly basis as well as before cryopreservation. You should also test any new cells you get from other labs or from commercial sources (if they have not been tested for mycoplasmas).
PCR Mycoplasma Test Kit I
- Detection of mycoplasmas with PCR Mycoplasma Test Kit I/C. Left to right: 100 bp DNA ladder; Positive control; 10; 100; 1,000; 10,000; 100,000 mycoplasmas/sample volume. Internal control DNA: 479 bp band; Positive sample/control: 270 bp band.
PromoKine's PCR Mycoplasma Test Kit I utilizes highly-sensitive PCR technology for fast, convenient and reliable in situ detection of contaminating mycoplasmas in various biological materials including cell cultures and virus stocks.
We provide qualitative as well as quantitative PCR assays:
- PCR Mycoplasma Test Kit I/C for the qualitative conventional PCR or
- PCR Mycoplasma Test Kit I/RT for the quantitative real-time PCR.
Advantages
- highly sensitive - detection of 10-15 fg of mycoplasma DNA that corresponds to 10-15 mycoplasmas per sample volume
- highly specific for a wide range mycoplasma species (including all mycoplasma species that are responsible for cell culture contamination)
- reliable - internal control DNA and positive control DNA optimize reproducibility and minimize operator-dependent variability of results
- convenient - contains optimized PCR reagents lyophilized in PCR tubes allowing convenient testing to be performed periodically or routinely for quality control
- user friendly - one protocol using standard lab equipment detects all relevant species. Allows for direct testing of culture stocks: elimination of antibiotics or special growth conditions are not required for testing.
- easy sample preparation - templates for PCR analysis are prepared by boiling the supernatant of cell cultures or other sample material.
- results are easily determined - a clear and easily interpretable result is obtained with one PCR run.
- rapid - the whole procedure can be completed in about 3 hours.
PCR Mycoplasma Test Kit I/C utilizes the polymerase chain reaction (PCR), which was established as the method of choice for highest sensitivity in the detection of mycoplasma and acholeplasma contamination in cell cultures and other cell culture derived biologicals. Detection requires as little as 10-15 fg of mycoplasma DNA corresponding to 10-15 mycoplasmas per sample volume. The primer set is specific for the highly conserved rRNA operon, or more specifically, the 16S-rRNA coding region in the mycoplasma genome. This allows for detection of M. orale, M. hyorhinis, M. arginini, M. fermentans, M. salivarium, M. hominis, usually encountered as contaminants in cell cultures, but also Acholeplasma laidlawii and many other species. The kit does not detect the clinically relevant M. pneumoniae and Ureaplasma urealyticum which are not known as cell culture contaminants. Eukaryotic and bacterial DNA is not amplified by using the kit. Only one protocol is needed for the detection of all mycoplasma species. The detection procedure can be performed within 3 hours. PCR Mycoplasma Test Kit I/C also provides internal control DNA (as negative control or for verifying a successful PCR run) as well as a positive control. When running the PCR with the internal control DNA, a successfully performed reaction is indicated by a 479 bp band on the agarose gel. The positive control yields a band at 270 bp.
All components required for the PCR (primer, nucleotides, control DNA) come lyophilized in PCR tubes except the Hot-Start Taq polymerase included in the kit. For setting up the PCR, simply cut off the number of tubes required, rehydrate with rehydration buffer (containing the Hot-Start Taq polymerase) and add the sample.
PCR Mycoplasma Test Kit I/RT
The PCR Mycoplasma Test Kit I/RT shows excellent sensitivity and specificity and allows to obtain reliable PCR results extremely fast (in "real time"), particularly for routine testing for mycoplasma contamination. It contains mycoplasma-specific probes and and comes with Inhibition Control DNA as well as Positive Control DNA. The kit detects a wide range of mycoplasma species and is compatible with most of the common Real-Time PCR instruments (e.g. Light Cycler® 1/2, SmartCycler® II, ABI Prism® and Stratagene Cyclers).
We provide two variants of this kit which are optimized for different Real-Time PCR instruments:
RT-PCR Instrument | Kit Variant A | Kit Variant B |
LightCycler® 1.2 | + | – |
LightCycler® 1.5 | + | – |
LightCycler® 2.0 | + | o |
LightCycler® 480 | + | o |
Rotorgene 3000 | + | o |
Rotorgene 6000 (5-plex) | + | o |
ABI Prism® 7000 | – | + |
ABI Prism® 7300 | – | + |
ABI Prism® 7500 | – | + |
ABI Prism® 7700 | – | + |
ABI Prism® 7900 | – | + |
iCycler iQ® | o | o |
iQ™5 | + | o |
Opticon 2 | – | o |
Chromo 4 | o | o |
Stratagene MX3000P/MX3005P | + | o |
Cepheid SmartCycler | + | o |
| AppliedBiosystems StepOne Plus™ | o | + |
+ = recommended Kit Variant
- = detection of internal control not possible
o = not tested so far, but compatibility is expected
Kit Components PCR Mycoplasma Test Kit I/C
- Test Reaction Tubes with lyophilized primer sets and nucleotides at optimized concentrations as well as internal control DNA to ensure accurate testing
- Positive Controls for easy result verification
- Rehydration Buffer
- Highly-active Hot-Start Taq DNA Polymerase
- Instruction manual
Kit Components PCR Mycoplasma Test Kit I/RT
- Primer&Probes: containing lyophylized primer set; nucleotides dATP, dCTP, dGTP and dUTP; mycoplasma specific probe; internal control probe for 25 reactions
- Inhibition Control Spike: containing artificial DNA fragments (lyophylized; prepared by PCR, non-infectious; for at least 25 reactions)
- Positive Control Spike, containing DNA-fragments of Mycoplasma orale (lyophylized; prepared by PCR, non-infectious; for at least 25 reactions)
- Reaction Buffer, 1x concentrated
- DNA-free Water
- Hot-start DNA Polymerase
- Instruction manual
PCR Mycoplasma Test Kit II
PromoKine's PCR Mycoplasma Test Kit II has also been designed to detect sensitively and specifically the presence of mycoplasmas that are contaminating biological materials such as cultured cells.
The kit includes a unique 5x master mix that contains all the ingredients required for PCR: nucleotides, primers, a high performance Taq Polymerase and magnesium. No prior preparations are required for PCR, other than the sample to be tested. The reaction mix contains a precipitant allowing for direct loading of PCR products onto an agarose gel. After performing agarose gel electrophoresis, positive samples will yield a 270 bp fragment. The test takes approximately five hours to obtain clear results and detects all mycoplasma commonly found in cell cultures.
The primers have been designed to detect those mycoplasma species responsible for most contaminations in cell cultures (including Acholeplasma) and were tested to be specific for mycoplasma DNA only, not reacting with animal or bacterial DNA.
Kit Components PCR Mycoplasma Test Kit II
- Reaction Mix with primer sets, nucleotides & Taq DNA Polymerase at optimized concentrations
- Buffer Solution
- Positive Template Control for easy result verification
- Comprehensive instruction manual
In sensitivity tests for the detection of defined mycoplasmas, the PromoKine Mycoplasma Test Kits I & II were found to be the most sensitive in comparison to other test kits currently available on the market. The ability to routinely perform rapid and simple tests to detect mycoplasma contamination in cell cultures facilitates the eradication or treatment of contaminated cells.
M. fermentans: 240 CFU/ml (using Mycoplasma Test Kit II)
M. capricolum: 110 CFU/ml (using Mycoplasma Test Kit II)
M. penetrans: 200 CFU/ml (using Mycoplasma Test Kit II)
M. hyorhinis: 210 CFU/ml (using Mycoplasma Test Kit II)
PCR Mycoplasma Test Kit III
- Detection of mycoplasmas with PCR Mycoplasma Test Kit III. Left to right: A. laidlawii, S. citri, M. synoviae, and M. orale (50 cfu/ml each); Cell culture supernatant (not infected); Negative control; Positive control; DNA size standard. Internal control DNA: 160 bp band; Positive sample/control: 260-270 bp band.
The new PCR Mycoplasma Test Kit III utilizes conventional PCR technology for convenient and highly sensitive detection of mycoplasma (detects ~50 cfu/sample volume). The primer set is specific for the highly conserved 16S-rRNA coding region in the mycoplasma genome. Thus, the kit reliably detects all mycoplasma commonly found in cell culture, including the Acholeplasma and Spiroplasma genera. The kit has been positively tested against all mycoplasma strains listed in the European Pharmacopoeia, but may detect even more mycoplasma species (~70-120 species). It does not detect microorganisms such as Streptococcus sp., Clostridium sp. and Lactobacillus sp. which are phylogenetically closely related.
The kit includes all components required for PCR (nucleotides, primers, a high performance Hot-Start Taq polymerase, and an agarose gel loading dye). Similar to the PCR Mycoplasma Test Kit II, all components are premixed at optimal concentrations in a ready-to-use and ready-to-load 1x master mix. The kit also contains positive control DNA for easy result verification. Like the PCR Mycoplasma Test Kit I (and in contrast to the PCR Mycoplasma Test Kit II) the PCR Mycoplasma Test Kit III also provides an internal control DNA to verify a successful PCR run. The test takes approximately 3 hours to run and obtain clear, reproducible results.
See also:
RTU DNA Ladders
Fluorescent Cell Stains also suited for mycoplasma detection (e.g. Hoechst 33258, DAPI)
See also: PromoCell's cell culture products - particularly cells and media for normal human cell culture. >More Information
Note: Mycoplasma safety is also topic of a training course of our PromoCell Academy! >More Information