Biological reagents

1mycoalert mycoplasma detection kitProduct features:

(1) Only 1 μ l of culture cell supernatant and a water bath are needed. Genomic DNA extraction is not required. Complex experimental equipment such as fluorescence microscope, PCR instrument, electrophoresis instrument, gel imager and bioluminescence detector are not required;

(2) A simple, fast, and sensitive cell mycoplasma detection method that only takes 1 hour to produce results;

(3) Only one step is needed to complete the entire experiment, and the experimental results can be easily recognized by the naked eye based on the color change of the reaction solution. No electrophoresis is required, avoiding the risk of false positives caused by multiple steps of opening the lid;

(4) The sensitivity is much higher than that of PCR method, and it can detect various common mycoplasma in cell culture (such as Mycoplasma hyorhinis, Mycoplasma hyorhinis, Mycoplasma oralis, Chlamydia cholerae, Mycoplasma fermentum, Mycoplasma salivarius, Mycoplasma hominis, and Mycoplasma pyriformis, accounting for 98% of mycoplasma contamination).

IImycoalert mycoplasma detection kitComposition (25T):

3、 Experimental steps:

(1) Preparation of supernatant for cell culture medium to be tested

When the test cells are cultured for 2-3 days and the melting degree is preferably above 80%, take a volume of 200 μ L or more of cell culture medium, centrifuge at 200g for 5 minutes, and then take the supernatant for subsequent testing (do not take 50 μ L of culture medium and cell sediment at the bottom of the tube);

(2) Reaction system preparation (first thaw and melt solution A, solution B, positive control, and paraffin oil, and immediately place them on ice)

Take out solution A from -20 ℃, let it thaw and melt, then gently invert it up and down and mix well. Prepare the following reaction system in a microcentrifuge tube based on the number of cell samples to be tested (the preparation process of the reaction system is very important on ice);

Note: Each experiment requires 1 negative control and 1 positive control. Due to the presence of pipetting errors, it is generally recommended to prepare N+3 of the above reaction systems for testing N samples

Gently mix with a shaker, then transfer to PCR tubes (recommended to use 200 μ l volume PCR tubes), with 24 μ l of the mixed reaction solution per tube;

(3) Sample loading (it is very important to keep the entire loading process on ice, especially the paraffin oil covering the reaction solution needs to be pre cooled on ice)

Sample to be tested: Add 1 μ l of the supernatant of the culture medium after centrifugation to the reaction tube, and then gently blow it up and down 5 times with this pipette;

Negative control: No sample added or 1 μ l sterile water added;

Positive control: Add 1 μ l of positive control sample and gently blow it up and down 5 times with this liquid gun;

Then gently add 25 μ l of paraffin oil above each reaction tube solution (in a water bath for reaction) to prevent inaccurate results due to liquid evaporation. Please pay attention to changing the nozzle each time when adding paraffin oil to prevent cross contamination between samples. If the reaction is carried out in a PCR instrument, paraffin oil does not need to be added.

(4) Reaction

Set the temperature of the water bath or PCR instrument to 61 ℃, place it in a reaction tube, and incubate for 60 minutes;

Remarks:

1. The actual temperature of the water bath may deviate from the set temperature. It is recommended to calibrate it with a thermometer first. In fact, reactions can be carried out at temperatures between 59-63 ℃, but it will affect the detection sensitivity;

2The reaction product cannot be opened, otherwise the generated aerosol may cause false positives in subsequent testing. It is recommended to wrap the reaction tube in a plastic bag or gloves, dispose of it in a dedicated garbage bin, and clean it up in a timely manner;

3. It is not recommended to use an oven or metal bath for heating reactions;

(5) Result judgment

After reacting at 61 ℃ for 60 minutes, immediately remove the reaction tube and place it at room temperature. Observe the reaction results in a well lit environment (it is recommended to use white paper as the background). If the reaction solution is still blue purple, it is judged as negative; If the reaction solution is sky blue, it is judged as positive.

4、 Related literature

1、ShiyuHe,YanzhiHuang,YanlingZhao. AReverseTranscription-PolymeraseSpiralReaction(RT-PSR)-BasedRapidCoxsackievirusA16DetectionMethodandItsApplicationintheClinicalDiagnosisofHand,Foot,andMouthDisease. FrontiersinMicrobiology.2020; 12(11):734-743.

2、AudreyJean,FlorenceTardy,OmranAllatif. AssessingmycoplasmacontaminationofcellculturesbyqPCRusingasetofuniversalprimerpairstargetinga1.5kbfragmentof16SrRNAgenes. PLOSONE.2017; 12(2):e0172358.

3、ZohreSoheily,MohammadSoleimani,KeivanMajidzadeh-Ardebili. DetectionofMycoplasmaContaminationofCellCulturebyALoop-MediatedIsothermalAmplifcationMethod. CellJ.2019; 21(1):43-48.

4、XinXu,XueyuWang,WenHu. AnImprovedPolymeraseCross-LinkingSpiralReactionAssayforRapidDiagnosticofCanineParvovirus2Infection. FrontiersinVeterinaryScience.2020; 7(57):1629-1636.