Explain why a mycoplasma PCR kit might give a negative result when u.pdf
- 1. Explain why a mycoplasma PCR kit might give a negative result when used to test for mycoplasma in a cell line containing mycoplasma Solution Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasma can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based methos, indirect staining and an agar and broth culture. These three tests for detecting Mycoplasma take one day to 3-4 weeks and such tests should be an obligatory component of quality control in every tissue culture laboratory. Mycoplasma refers to any of over 200 species of tiny bacteria that invade cell cultures and may even persist unrecognized for some time. They can come from sources as mundane as other cells introduced to lab via media, sera or lab personnel. So, it is important to test your cell lines for mycoplasma regularly. Consequences to your cells of mycoplasma infestation include changes in growth rates, gene expression, morphology, metabolism and viability. Even though most lab contamination can be blamed on as few as eight main culprit species, these differ enough that no one test can find them all. And the little blighters are hardy – most antibiotics used in cell culture are ineffective against mycoplasmas. For example, mycoplasmas lack a cell wall, which is the key to penicillin’s success. Flawless cell culture technique is one of the best preventive tools and testing to ascertain which cultures are clean and which cultures are infected – so you can take quick and aggressive action before infestation spreads. The M.hyorhinis strain is picked up by the PCR method of detection. This is the quickest but also the least sensitive, mycoplasma assay. In this method, you perform PCR on samples of your suspected cell cultures, using primers specific for mycoplasma DNA, usually mycoplasma’s 16S rRNA genes. Running the PCR product on a gel shows presence of mycoplasma DNA by bands of distinct sizes. One major limitation of PCR is that prior information about target sequence is necessary in order to generate the primers that will allow its selective amplification. This means that, typically, PCR users must know precise sequences upstream of target region on each of the two single-stranded templates in order to ensure that DNA polymerase properly binds to primer-template hybrids and subsequently generates entire target region during DNA synthesis. Like all enzymes, DNA
- 2. polymerases are also prone to error, which in turn causes mutations in PCR fragments that are generated. Specificity of PCR fragments can mutate to template DNA, due to nonspecific binding of primers. Some important general aspects should be considered when performing PCR technique: 1) Sensitivity of the procedure makes it susceptible to contaminations with target DNA which is present in high amounts after first amplification of mycoplasma-specific DNA. Thus, extreme care has to be taken to prevent carry-over of target DNA fragments. This is especially the case when a nested PCR is performed. 2) The PCR should be performed with extracted DNA and not with a crude lysate of cell culture supernatant because cell culture components may contain inhibitors of Taq polymerase. 3) Use of antibiotics in cell culture should be minimized and the cell cultures should be cultured without antibiotics for many passages or at least two weeks to allow the mycoplasmas to grow to detectable amounts or to ensure that no residual mycoplasmal DNA is left in culture medium. 4) A positive result of the PCR does not necessarily indicate viable contaminants, especially after a mycoplasma elimination procedure using antibiotics against mollicutes. So, PCR method should be properly established and all assays must be performed with the utmost care. The specificity of primers should be narrow enough to exclude amplification of sequences from other common bacteria, which may be contaminants of the PCR reagents. The PCR can be performed with a single round of amplification or as nested PCR with two primer pairs. Second method increases sensitivity and the specificity. But one of the drawbacks of nested PCR is the possible generation of false positive results due to contamination with target DNA. For routine cell culture technology, the PCR is satisfactory to detect mycoplasma contaminations because the titer of the mycoplasmas in cell cultures is sufficiently high to be detected by the PCR. Special conditions, e.g., after mycoplasma elimination procedures or for detection of mycoplasma in cell culture products like FBS, the nested PCR might be of advantage. Another possibility to increase the sensitivity of the assay is to perform a reverse transcription PCR to detect ribosomal RNA which is more abundant in the cells than the rRNA-coding DNA. But, the latter option is clearly more labour-intensive. In summary, one would suggest to perform a single PCR with genomic DNA for routine cell culture and to test the cultures frequently for contaminations.