Mycoplasma is the smallest prokaryotic microorganism, only 0.1-0.3 μm in size. Due to their small size, mycoplasmas can penetrate rated filters (0.22~0.45 μm). Mycoplasma contamination remains a major problem in cell culture. Mycoplasmas can alter the DNA, RNA, and protein synthesis of culture cells, but they may not noticeably affect cell growth rates in many cases. Therefore, it is difficult to discover mycoplasma contamination with the naked eye. And mycoplasma detection should be performed regularly during cell culture.

There are many methods to detect mycoplasma contamination, such as the culture-based method, fluorescent staining method, ELISA and so on. However, most of these methods are time-consuming, complex to operate, and not highly sensitive. PCR-based detection is a relatively simple method within a few hours by PCR and electrophoresis. If necessary, PCR products can be sequenced to determine the species of contaminated mycoplasma.

The PCR Mycoplasma Detection Kit is a kit for the specific detection of mycoplasma by nested PCR. Nested PCR uses two pairs of primers to amplify the mycoplasma genome DNA, which is more sensitive than one-step PCR detection. As shown in Figure 1, the rRNA gene sequence of prokaryotes is very conserved, and the rRNA operon encodes the DNA of rRNA Space regions vary greatly between various biological species, such as the interval between 16S and 23S. Thus, a pair of F1/R1 primers can be designed on the conserved region DNA encoding 16S and 23S and can be used for amplification, which is the first round of PCR of nested PCR (1st PCR). 1st PCR can be used to initially determine whether there is mycoplasma contamination; An F2 primer designed on the conserved region of the DNA spacer region encoding 16S and 23S rRNA, and an R2 primer designed on the DNA encoding 23S rRNA are used for the second amplification of PCR (2nd PCR) for further confirmation. Compared with one-step PCR, nested PCR can greatly improve the specificity and sensitivity of detection.

The kit provides a rapid, efficient, and highly sensitive detection method for mycoplasma contamination, and Positive Control is provided to confirm that the kit is properly tested and whether the sample contains PCR inhibitors. If mycoplasma contamination is found after detection, the contaminated cells can be discarded directly. If cells are precious, consider treating the cells with Mycoplasma Remover Reagent.

Figure 1: Principle of nested PCR-based mycoplasma detection

Figure 2: Schematic of agarose gel electrophoresis of 1st PCR and 2nd PCR products.
1, 2, 3 are 1st PCR products; 1#, 2#, 3# are the 2nd PCR products. The templates for each lane are: 1 and 1#, negative cell supernatant; 2 and 2#, mycoplasma contaminated cell supernatant; 3 and 3#, Positive Control.