Description

Mycoplasma, the smallest and self-replicating prokaryotes, are common contaminant of primary and continuous cell line cultures. It has been shown that micoplasma contamination affects cell growth, morphology and metabolism. Because of their small size and flexibility, mycoplasma can pass through commonly used filters of 0.2 μm. Furthermore, unlike bacterial and fungi, micoplasma contamination is resistant to commonly used antibiotics, and not easy to be spotted visually. These drawbacks emphasize the need for regular screening in control of mycoplasma contamination. Conventional screening methods include microbiological culture, fluorescent DNA staining and biochemical detection methods. However, mycoplasma culture is restricted to specialized laboratories and takes 2-4 weeks. Results of DNA fluorochrome staining are difficult to interpret due to the presence of broken nuclei. Various biochemical detections are time consuming and often lack of sensitivity. Recently, polymerase chain reaction (PCR) based methods have been developed as a quick and convenient detection of low level mycoplasma infection, which offers great advantage over the conventional methods because of its extreme sensitivity and specificity.

Computer alignment studies of mycoplasmal 16S rRNA sequences have revealed the existence of highly conserved sequences. Primers designed according to these sequences allow the specific amplification of mycoplasma DNA fragment and thus highly sensitive detection of mycoplasma. ScienCell™ Mycoplasma PCR Detection Kit is a 16S rRNA-based PCR assay including 2X reaction buffer, primer set, as well as internal amplification control (IAC) and positive sample control. The genus-specific primer set we select recognizes the five typical contaminating mycoplasma species (i.e. M. hyorhinis, M. arginini, M. orale, M. fermentans, and A. laidlawi), which account for 98% of contaminations, as well as members of the genera Ureaplasma, Spiroplasma, and Acholeplasma, which account for the remaining 2% of contaminations. Eukaryotic and bacterial DNA is not amplified by this kit. The IAC is designed to be co-amplified simultaneously with the target mycoplasma sequence by the same set of primers. The resulted control band, which is distinguished from the target band by a difference in molecular mass, indicates the occurrence of DNA amplification. Therefore, false negative results due to the presence of PCR inhibitors in some cell cultures can be identified. Our kit also provides a positive sample control, which is noninfectious genomic DNA (gDNA) of M. fermentans. On the other hand, a no template (i.e. DNA-free DI H2O) negative sample control can help to determine whether there is a false positive result due to carry-over contamination. Each experiment should include both positive and negative sample controls, as well as the IAC.

Product Use

This assay kit is used to detect mycoplasma in vitro. It is for research use only. Not for use in animals, humans, or diagnostic procedures.