Detection of Mycoplasma with PCR

Why is the PCR better than other traditional methods?
With PCR testing, results are obtained within a few hours, since the presence of contaminant mycoplasma can be easily detected by verifying the bands of amplified DNA fragments in electrophoresis. Furthermore by sequencing the PCR product the mycoplasmas can be determined to the species.

Is a reamplifaction or a “Nested –PCR” better?
The sensitivity and also the specificity can be increased by a nested PCR where the primer pair of the following PCR is located within the first amplified DNA sequence. A simple reamplification is not recommended because the resulted DNA smear can complicate the analysis.  

Can the PCR distinguish between the DNA of viable cells and the DNA of destroyed cells?
The classical PCR can only amplify DNA. There are no internal mechanism of the PCR method to determine if the DNA comes from a viable cell or from a cell which was killed by treatment with antibiotic or by autoclave sterilisation. The genomic DNA of the destroyed cells is in the surrounding liquid and it is also detectable. This is one of the major problems or disadvantages of the method.

Is it possible to digest the genomic DNA from destroyed cells with DNAse?
If cells die after treatment with antibiotics or sterilisation the DNA is released into the surrounding medium. The DNAse treatment of released genomic DNA which comes from non-viable cells is the best way to remove the contaminating DNA. Furthermore it is also possible to digest the total DNA and extract the RNA. The RNA is normally only detected  in viable cells because active cellular RNAses degrade RNA from lysed cells.

How should I perform a PCR which gives reliable and significant results to detect only viable mycoplasmas?
The special preparation of the samples is important. Aliqouts from the supernatant of the medium and the cells are taken. The samples are centrifuged at low speed and the pellets are washed briefly,  three times using fresh media or PBS. The samples are treated with diluted DNAse and the DNA is then extracted by lysis of the cells with a chaotropic solution (part of the DNA- or RNA extraction kit). Optionally the whole genomic DNA of the cells is digested with the DNAse and the RNA is extracted. The RNA must be reversed transcribed in the first step and amplified by PCR (RT-PCR). In both cases the resulting DNA is amplified by PCR. The PCR products are analysed by gel electrophoresis and compared with the positive controls.

How to prepare a positive control for PCR?
In general, most of the commercially available Mycoplasma detection kits contain the positive control DNA which contains a defined number of target sequence copies. The control sequence can be obtained by amplification of a DNA of a mycoplasma strain (ATTC or DSM). The strains are delivered as a lyophilisate. Enrichment by cell cultivation is unnecessary as enough template DNA for PCR amplification is available. The PCR products are cloned into a T-vector. By serial dilution (1:10, 1:100,...) of the known DNA concentration, the highest dilution can be used as a sensitivity control. If the PCR can detect the highly diluted control the PCR conditions are suitable.

Are there measures to prevent cross contaminations?
To prevent false positive results by cross contamination the preparation area for sample DNA must be different to that of where the PCR controls are prepared (aerosols can transmit DNA target sequences). Furthermore all pipette tips used should contain filters. All the buffers, primers, nucleotides and the enzyme should be prepared as a ready and cooled master mix. Prior start of the PCR the target sequences are added. The negative control, which contains all reagents except the target sequence, gives information if there is cross contamination. After PCR analysis the negative control must be negative.