Mycoplasma Detection Set

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:
        High Sensitivity: Sensitive enough to detect trace amount mycoplasma contamination in cell culture medium.
        Simplicity: Only cell culture medium required and no DNA preparation and cell collection.
        Broad Detection Range: Detects common strains of Mycoplasma with a simple protocol.
        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.

Electrophoresis:
Take 10 L of PCR products to run 1.5% agarose gel.

Table 1: PCR Schemes

1st Round PCR        Volume                PCR program
Mycoplex I (2X)        10 L               
Step 1: 94°C        30"
Step 2: 94°C        30"
Step 3: 55°C        45"
Step 4: 72°C        45"

Repeat step 2-4 for 35 cycles

Step 5: 72°C        1'
MacTaq        0.5 L               
Template (culture medium or 2 L positive control)        2 L               
Water (nuclease-free)        7.5 L               
                       
2nd Round PCR                       
Mycoplex II (2X)        10 L               
MacTaq        0.5 L               
Template (1st Round PCR product)        0.5 L               
Water (nuclease-free)        9 L               
       

Table 2. Size of PCR product amplified from most common mycoplasma species (in bp)

Species                1st PCR*        2nd PCR                Notes
M.hyopneumoniae        肺炎支原体        681        237               


* 1st round band sometimes does not show up on agarose gel when using culture media instead of genomic DNA as template, especially when mycoplasma contamination is slight.

M.neurolyticum        溶神经支原体        501        196               
M.fermentans        发酵支原体        491        195               
M.pulmonis        肺支原体        477        189               
M.hyorhinis        猪鼻支原体        448        211               
M.orale        口腔支原体        423        179               
M.capricolum        山羊支原体        415        179               
M.arthritidis        关节炎支原体        408        157               
M.salivarium        唾液支原体        403        151               
M.hominis        人型支原体        370        148               
M.arginini        精氨酸支原体        369        145               
M.urealyticum        解脲支原体        482        154