Primer set and kit for detecting mycoplasma, and method for detecting mycoplasma pollution

A mycoplasma and kit technology, which is applied in the biological field, can solve the problems of low reliability of research results, insufficient broad-spectrum, insufficient sensitivity, etc., and achieve the effects of easy interpretation, good broad-spectrum and broad application prospects.

Inactive Publication Date: 2016-12-21
SHANGHAI XP BIOMED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For relevant scientific researchers, the most frightening thing is that mycoplasma pollution changes many parameters unconsciously, which greatly reduces the credibility of research results
[0003] Commonly used detection methods for mycoplasma include traditional culture method, ELISA method, PCR method, etc. At present, PCR method is the mainstream detection method for mycoplasma. Not enough or not specific enough, etc.

Method used

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  • Primer set and kit for detecting mycoplasma, and method for detecting mycoplasma pollution
  • Primer set and kit for detecting mycoplasma, and method for detecting mycoplasma pollution
  • Primer set and kit for detecting mycoplasma, and method for detecting mycoplasma pollution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Design and synthesis of primer sets

[0035] First, select the mycoplasma species to be detected: According to the most common mycoplasma contamination cases in the cell culture process, 15 common mycoplasma species are selected as the target mycoplasma species to be detected: M.fermentans, M.hyorhinis, M.arginini , M. orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. bovis, M. pneumoniae, M. pirum, M. capricolum, Acholeplasma or Spiroplasma;

[0036] Then, compare the gene bank: search from the American gene database Genbank, compare the 16S rDNA sequences of the above 15 target mycoplasma species, use xxxx software to analyze and compare homologous sequences, and find a conserved and specific sequence as the target fragment;

[0037] Subsequently, the primers required for the PCR amplification reaction were designed with the above-mentioned target fragments.

[0038] Finally, test the specificity and sensitivity of the designed primers; i...

Embodiment 2

[0043] Embodiment 2: the preparation of kit

[0044] 1) Synthesize the primer set with reference to the above-mentioned Example 1;

[0045] 2) Mycoplasma lysis buffer: mycoplasma lysis reagent (product number 07066) purchased from SIGMA-ALDRICH company;

[0046] 3) Polymerase chain reaction reagents: purchased from the "low background, high sensitivity PCR series" products produced by TaKaRa Biotechnology Company, mainly including: Taq enzyme, PCR reaction buffer, MgCl 2 and dNTPs;

[0047] 4) Positive control: a plasmid containing the conserved sequence of mycoplasma 16S rDNA (270bp);

[0048] 5) Negative control: sterile water.

[0049] The forward sequence and reverse sequence of the above primer set were placed in airtight containers; the positive control and negative control were respectively placed in airtight containers; and then assembled into the kit together with the purchased polymerase chain reaction reagent products.

[0050] The specificity evaluation of ...

Embodiment 3

[0062] Embodiment 3: detect mycoplasma pollution

[0063] Step 1) Mycoplasma lysis: use the mycoplasma lysis buffer in the kit of Example 2 to process the samples to be tested (samples 1, 2 and 3) to obtain a lysate;

[0064] First, take 0.5-1 ml of the sample to be tested (cell culture supernatant) and add it to a 2 ml centrifuge tube; briefly centrifuge at 500 rpm for 60 seconds to pellet cell debris; transfer the centrifuged supernatant into a new In a sterilized 2 ml centrifuge tube; centrifuge at 20000rpm for 10 minutes to precipitate mycoplasma, then gently aspirate and discard the supernatant; then add 50 microliters of mycoplasma lysis buffer to the centrifuge tube, gently pipette Swipe to mix the precipitate, and heat at 95 degrees Celsius for 3 minutes to obtain a lysate containing mycoplasma DNA;

[0065] Step 2) PCR amplification: mixing the lysate obtained in step 1) with the polymerase chain reaction reagent and the primer set to perform a PCR amplification re...

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Abstract

The present invention discloses a primer set for detecting mycoplasma. The primer set comprises a forward primer having a sequence represented by SEQ ID No.1; and a reverse primer having a sequence represented by SEQ ID NO.2. The invention further discloses a kit for detecting the mycoplasma by using the primer set, uses of the kit in detection of mycoplasma pollution, and a detection method for detecting the mycoplasma pollution by using the kit. The kit of the present invention has advantages of simple operation, broad spectrum, strong specificity and high sensitivity, can rapidly detect the mycoplasma pollution, and has wide application prospect.

Description

technical field [0001] The invention relates to a primer set, a kit and a method for detecting mycoplasma contamination for detecting mycoplasma, belonging to the field of biotechnology. Background technique [0002] Mycoplasma (Mycoplasma) pollution is one of the most common pollutions in the cell culture process, especially in the culture process of mammalian cells, the problem of mycoplasma pollution is particularly troublesome. The main reasons are the following three points: 1) Mycoplasma is very small in size, The diameter is 0.1 μm, which cannot be removed by general filter sterilization, and its morphological structure is difficult to see under an optical microscope; 2) Mycoplasma is tenacious, can survive in alkaline conditions (pH 7.6-8.0), and is resistant to penicillin; 3) At the initial stage of cells being contaminated by mycoplasma, some sensitive cells can see that the growth and proliferation of the cells slow down and the shape becomes round. However, most...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/35
CPCC12Q1/689C12Q1/686C12Q2565/125
Inventor 张智培熊延狮
Owner SHANGHAI XP BIOMED
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