Storage and Stability:

The product is shipped at 4 ℃. Store all the components at -20 ℃ upon receipt. Under these conditions, the product is stable for at least two years.

Introduction:

One-step Quickcolor Mycoplasma Screening Kit is designed to detect mycoplasma in cell culture media or other liquid sample. Mycoplasma contamination is a major headache for the cell culturist. Mycoplasmas are small, simple bacteria which lack a cell wall and represent one of the most prevalent and serious sources of cell line contamination. Mycoplasma contamination is invisible with no discernible change in turbidity or pH even at densities as high as 107-9 cells/mL.

In fact the most common source of mycoplasma infection in cell culture research is another previously infected culture brought into the laboratory. Another major source is the laboratory worker as human mycoplasma continues to be a major source of mycoplasma infection in cell culture.

Mycoplasma are a problem because they can induce changes to the cell cultures which include altered growth rates, morphological changes, chromosomal aberrations, and altered cell metabolism. In fact, a mycoplasma contaminated cell line due to the alteration of its characteristics can be regarded as a different cell line.

Regular testing for mycoplasma contamination is recommended for all laboratories carrying out cell culture. There are a number of mycoplasma tests available, each with its own advantages and disadvantages. A combination of three methods (culture test, DNA stain and PCR) is recommended for routine Screening , ensuring strains of mycoplasma which do not grow in vitro, can be detected by DNA stain and PCR.

The culture test is most sensitive, detecting nearly all species. However, one of the major mycoplasma species present in the cell culture media, M. hyorhinis, is not detectable by the culture method. In addition, it takes a month to obtain the result, the medium required is complex with a relatively short shelf-life and live positive controls must be included.

Hoechst stain is a general DNA stain, not specific to mycoplasma. But interpretation of the results can be problematical.

PCR provides a quick, relatively high through method for detecting mycoplasma. It is both relatively cost effective and requires minimal training.

Working with authenticated cell lines free from mycoplasma contamination is a prerequisite for the generation of robust, reliable, and reproducible data in the biomedical research field. As of mid-2013, manuscripts submitted to Nature journals must be accompanied by a reproducibility checklist, one of which requires researchers to provide proof of testing for mycoplasma contamination.

One-step Quickcolor Mycoplasma Screening Kit is easy and fast, and the results can be obtained within one hour. Based on a special isothermal RCA (Rolling Circle Amplification), the reaction can be carried out even in a water bath. Positive reaction can be easily indicated by color change from pink to blue-green, which can be judged by the naked eye.

This kit allows Screening of various mycoplasma species (such as M. Hyorhinis，M. Fermentans，M. Arginini，M. Hominis，M. Orale，M. Salivarium，M. Pirum，Acholeplasma Laidlawii，M. Agalactiae，M. Bovis，M. Buccale，M. Arthritidis，M. Pulmonis ) with extremely high sensitivity and specificity. The above seven mycoplasma species account for over 98% mycoplasma contamination in cell culture.

Kit Components:

Solution 1: 1150 μL (50 Test)
Solution 2: 50 μL (50 Test)
Boiled Mycoplasma DNA: 50 μL
Mineral oil: 1500 μL

Procedure:

1.Sample preparation:

Cell lines should be pre-cultured at least for 3 days before Screening . Samples should be derived from cultures that are at 70–90 % confluence. Cell culture supernatants can be tested directly.

2.Reaction system setup:

(1) Total reaction volume is 25 μL. It is recommended to perform a positive and a negative control reaction. Thaw Solution 1 at room temperature or at 37 ℃ and mix well. Solution 2 should be always stored at -20 ℃. Setup the reaction system according to Table 1.

Table 1. Reaction system

	Volume of Single Sample	Number of Samples	Total Volume
Solution 1	23 μL	N	23×N×1.08 μL
Solution 2	1 μL	N	1×N×1.08 μL

(2)Mix by pipetting several times and add 24 μL to each 0.2 mL PCR tubes.

(3)Add 1 μL mycoplasma DNA into the positive control tube and 1 μL cell culture supernatants into the test tube.

1.Reaction:

Select one of the following reaction methods

Reaction on a water bath. Add 25 μL mineral oil to each tube to prevent water evaporation and place the tubes in a water-bath at 61°C for 60 minutes.
Reaction on a thermal cycler PCR machine. Due to the great temperature variation of a general water-bath, if a PCR machine is available, please carry out the reaction in a PCR machine. Adding mineral oil to each tube is not necessary if the lid of PCR machine can be set at 100 °C. Set the PCR machine as following: 61 ℃，60 min；10 ℃, ∞；Lid, 100 ℃.

2. Results analysis: After reaction, if the solution color of the reaction tube is purple- red, this indicates that there is no mycoplasma present in the test sample. If the solution color of the reaction tube is blue-green, this indicates that the test sample is contaminated by mycoplasmas (Figure 1).

Figure 1. Results analysis by color change.

Note:

If the reaction is carried out in a water-bath, please make sure the temperature is accurate with mercury thermometer.
The reaction time at 61°C should be no more than 65 minutes.
After reaction, please don’t open the reaction tubes.
For Research Use Only. Not for use in diagnostic procedures

Ordering Information

Mycoplasma Elimination Products：Mycokiller-1(E201) ,Mycokiller-2(E202) and LabCleaner Powder(C301).

One-step Quickcolor Mycoplasma Screening Kit - High Sensitivity Cell Culture Analysis