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PCR (Polymerase Chain Reaction) detection method of mycoplasma

A detection method and technology for mycoplasma, applied in the field of biological testing, can solve the problems affecting the physiological activity and influence of cultured cells, and achieve the effects of short detection period, strong result consistency and high stability

Inactive Publication Date: 2011-11-16
JINYUBAOLING BIO PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mycoplasma contamination significantly affects the physiological activity of cultured cells, which greatly affects the production of vaccines based on cell culture.
[0004] The traditional detection method is the culture method, which uses a nutrient-rich medium to directly culture the cells and serum to be tested for mycoplasma, and observe the formation of mycoplasma colonies on the medium, but the test period of this method is at least 3 weeks

Method used

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  • PCR (Polymerase Chain Reaction) detection method of mycoplasma
  • PCR (Polymerase Chain Reaction) detection method of mycoplasma
  • PCR (Polymerase Chain Reaction) detection method of mycoplasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the mycoplasma PCR detection of various experimental samples in FMDV vaccine production

[0029] 1 Sample preparation

[0030] Take 1ml of each of the following samples respectively, and transfer them to sterilized 1.5ml centrifuge tubes for later use.

[0031] 1) No. 1 BHK21 cell culture;

[0032] 2) No. 2 BHK21 cell culture;

[0033] 3) No. 1 FMDV virus solution;

[0034] 4) No. 2 FMDV virus solution;

[0035] 5) No. 1 nutrient solution for cell culture;

[0036] 6) Positive control, i.e. Mycoplasma hyorhini culture;

[0037] 7) Blank control, namely sterilized water;

[0038] 2 Sample Mycoplasma Nucleic Acid Preparation

[0039] The nucleic acid extraction method is:

[0040] 1) Put 1mL of the sample to be tested into a 1.5mL sterile centrifuge tube and centrifuge at 12000rpm for 20 minutes;

[0041] 2) Discard the supernatant, and dissolve the precipitate with 20 μl sterile ultrapure water;

[0042] 3) Put the centrifuge tube in a boiling wat...

Embodiment 2

[0070] Embodiment 2: mycoplasma PCR detection detection sensitivity experiment

[0071] 1. Sample Preparation

[0072] Count Mycoplasma to 10 8 BHK21 cell culture pollutants per ml were diluted 10 times, and 1 ml of each dilution was taken as a sample for later use.

[0073] 2. Experimental steps

[0074] The experimental procedure is the same as "Example 1".

[0075] 3. Results Observation

[0076] Electrophoresis results, as attached figure 2 As shown, the spotting sequence is as follows:

[0077] 1: DL1000 DNA Marker;

[0078] 2:10 -1 Dilute cell culture contaminants;

[0079] 3:10 -2 Dilute cell culture contaminants;

[0080] 4:10 -3 Dilute cell culture contaminants;

[0081] 5:10 -4 Dilute cell culture contaminants;

[0082] 6:10 -5 Dilute cell culture contaminants;

[0083] 7:10 -6 Dilute cell culture contaminants;

[0084] 8:10 -7 Dilute cell culture contaminants;

[0085] 9:10 -8 Dilute cell culture contaminants;

[0086] 10: Blank control, steri...

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Abstract

The invention relates to a PCR (Polymerase Chain Reaction) method for detecting mycoplasma, belonging to the technical field of biological detection. In the invention, PCR amplification and electrophoresis are carried out by extracting mycoplasma nucleic acids and applying mycoplasma specific primers to judge whether mycoplasma contamination exists in a detection sample or not, wherein if 435bp specific strips appear in an electrophoretogram, the result shows that the mycoplasma exists in the sample, and if 435bp specific strips do not appear, the result shows that the mycoplasma does not exist in the sample. By the detection and verification, the detection system provided by the invention has the characteristics of high specificity, good stability, short checking period and the like.

Description

technical field [0001] The invention belongs to the technical field of biological testing and relates to a PCR detection method for mycoplasma. Background technique [0002] Mycoplasma is a kind of prokaryotic microorganism lacking cell wall. Its size is generally between 0.2-0.3 μm, and it is highly pleomorphic. It has various forms such as spherical, rod-shaped, filamentous, and branched. About 1% of mycoplasma can pass through the bacteria filter. It is not easy to be colored by ordinary staining method, and it is very light by Giemsa staining, so it is not easy to be found under ordinary optical microscope. So far, more than 30 species of mycoplasma have been discovered. The common types of contamination in cell culture include: Mycoplasma arginini, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma fermentans ), Acholeplasma laidlawii, etc. [0003] Cells are an important tool commonly used in scientific research and production in the field of veterinary biological pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张贵刚刘国英郭伟徐师军张领月
Owner JINYUBAOLING BIO PHARM CO LTD
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