In cell culture, especially in passage cell culture, the mycoplasma contamination rate reaches 15-35%. Infection by mycoplasma can cause severe changes in cell function and gene expression and cause persistent damage. Conventional detection of mycoplasma is important because mycoplasma contamination can seriously affect the reliability, repeatability, and consistency of experimental results.
The traditional method of detecting mycoplasma is time consuming, usually takes one day to several weeks, and the operation is cumbersome and the accuracy is not high.
There are two standard methods for detecting mycoplasma given by the Chinese Pharmacopoeia: culture method and DNA staining method. The culture method is simple, the false positive rate is low, but the sample needs to be high, and it takes about 20 days, and the time is long. The results of DNA staining are intuitive, and the false positive rate is low, but fluorescence microscopy equipment is required, and the experimental operation is complicated.
Can you find a time-saving, labor-saving and accurate method to detect mycoplasma contamination?
At present, there are a variety of rapid detection kits for mycoplasma on the market. We have selected two common brands and compared their detection principles and operations.
First, the United States CLARK Bioscience brand one-step test kit
One-step Quickcolor Mycoplasma Detection Kit, developed by CLARK Bioscience, USA, uses isothermal amplification to detect genes of seven common mycoplasmas.
The detection method is similar to the PCR method. During the detection process, only a small amount of cell culture supernatant is needed, and a water bath or a constant temperature metal bath capable of controlling the temperature can be directly observed by the naked eye, and the whole process needs to be performed. About 1 hour.
The method has lower requirements on the instrument and theoretically has higher sensitivity. The disadvantage is that the number of mycoplasma detected is too small. Nearly 200 kinds of mycoplasma that have been discovered so far can only detect 7 of them. The detection range is too narrow and does not meet the requirements of the National Pharmacopoeia. Moreover, the results of the naked eye observation are subjective. If the experimenter has a problem of color blindness or color weakness, this method is best to give up.
Second, the United States Lonza brand bioluminescence detection kit
US Lonza MycoAlert TM brand and upgrade MycoAlert TM PLUS Mycoplasma Detection Kit, using the bioluminescence method, detecting mycoplasma activity. The mycoplasma enzymes tested are widely present in more than 180 mycoplasmas and are not present in eukaryotic cells.
This method utilizes the specific activity of the mycoplasma enzyme for selective biochemical detection. When the mycoplasma is dissolved, the released enzyme catalyzes a reaction with the substrate provided in the Mycoplasma detection kit, converts ADP to ATP, participates in the bioluminescent reaction, and can be measured by a spectrophotometer. If the sample is added after the substrate is added, the reading is increased, indicating the presence of mycoplasma contamination. The entire experiment takes about 20 minutes.
The method can detect the types of mycoplasma, and the detection results are quantified, easy to judge, and time-consuming. The disadvantage is that you need to configure the illuminator or multi-function microplate reader, the visibility is not as good as the amplification method, and the sensitivity may be lower than the amplification method.
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