Description:

PCR-based detection of mycoplasma contamination of cell culture.

Mycoplasma is a common and serious contaminant of cell cultures. It has been shown that more than 30% of cell cultures in the laboratory are infected with Mycoplasma. In continuous cell cultures, contaminating Mycoplasma may grow slowly without killing the cells but affecting various parameters including altered cellular proliferation and viability, morphological changes, cell transformation, mimicking virus infection, and inresponsiveness to drug treatment, etc., and ultimately leading to unreliable results. Mycoplasma detection is an important and necessary quality control measure.

Many of the testing procedures have been developed, which include DNA staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and PCR-based ELISA.  M&C Gene Technology provide our research community with reliable reagents and simple protocol, which allow for rapid and highly reproducible detection of mycoplasma contamination.

The primers used in this kit anneal to conserved regions of the Mycoplasma genome, allowing detection of the most common species of Mycoplasma (including M. opalescens, M. arginini; M. fermentans; M. caviae, M. hyorhinis, M. indiense, M. orale, Acholeplasma laidlawii and many more – see table 2 below).

Important Features:

l          High Sensitivity: Sensitive enough to detect trace amount mycoplasma contamination in cell culture medium.

l          Simplicity: Only cell culture medium required and no DNA preparation and cell collection.

l          Broad Detection Range: Detects common strains of Mycoplasma with a simple protocol.

l          Species Determination: The species of mycoplasma can be determined by sequencing the amplified products.

Procedures:

Preparation of template:

Culture cells for at least 3 days to reach >50% confluency. 2ul cell culture medium will be used as PCR template in a standard 20ul PCR reaction.

PCR Reaction:

Set up PCR reactions by following the schemes in Table 1. For heavy contamination, only the first round PCR reaction is required; for slight contamination, the second round PCR reaction will give more sensitive measure by amplifying the PCR product from the first round PCR reaction.