Products

Mycoplasma Test Kits

PCR kits for sensitive and rapid detection of mycoplasmas in cell culture

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.

 

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. Hoechst 33258)
  • DNA hybridization
  • Antibodies for specific mycoplasma species
  • Electronic microscope
  • PCR: specific primers

 

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

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.