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Mycoplasma contamination detection method and application

A mycoplasma and to-be-detected technology, applied in the biological field, can solve the problems of high price of mycoplasma detection kits, affecting the accuracy of results, and high use costs

Inactive Publication Date: 2018-08-03
SUZHOU GENDX BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the ATP enzyme method mainly depends on the activity of mycoplasma enzymes, any influence on the enzyme activity may affect the accuracy of the results. In 2015, Vahid Molla Kazemiha et al. found that the sensitivity and accuracy of the ATP enzyme method were lower than that of real-time fluorescent quantitative PCR. Law [9] , the ATP enzyme method needs to use a fluorescence spectrophotometer, and the commercialized mycoplasma detection kit is expensive, and the cost of large-scale promotion and use is relatively high

Method used

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  • Mycoplasma contamination detection method and application
  • Mycoplasma contamination detection method and application
  • Mycoplasma contamination detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] The 23 kinds of mycoplasma obtained from the above screening and plasmid synthesis were selected as detection targets,

[0108] The sequences of the three upstream primers are: 5'-ACCACCCGGTAAGGCTAAATACTAACCAGAC-3' (Seq ID No.1) can amplify M.bovis, M.bovoculi, M.fermentans, M.synoviae, M.pulmonis, M.flocculare, M. hyorhinis, M.hominis, M.arginini, M.arthritidis, M.hyosynoviae, M.orale, M.salivarium, M.hyopneumoniae, Spiroplasma citri a total of 15 kinds of mycoplasma; 5'-AGGACCATCTCCTAAGGCTAAATACTACTTGG-3'(Seq ID No.2 ) can amplify Acholeplamasalaidlaawii; 5'-CAAACCATTGGGTAAGCCTAAATACTAGTCAG-3' (Seq ID No.3) can amplify M.gallisepticum, M.pirum, M.pneumoniae, M.penetrans, M.genitalium, Ureaplasmaurealyticum, M.capricolum A total of 7 kinds of mycoplasma.

[0109] The sequences of the two downstream primers are: 5'-CACCCATTAACGGGCTCTGACTAATTGTAA-3' (Seq ID No.4) can amplify M.bovis, M.bovoculi, M.fermentans, M.synoviae, M.pulmonis, M.flocculare, M. .hyorhinis, M.homin...

Embodiment 2

[0133] Select the primers and probe sequences designed in Example 1, carry out the amplification test of the method version of the recombinase polymerase amplification (in conjunction with endonuclease IV), and construct the amplification reaction system as follows:

[0134] 30mM tris-acetic acid buffer pH8.0

[0135] 100mM potassium acetate

[0136] 14mM magnesium acetate

[0137] 3mM Dithiothreitol

[0138] 5% polyethylene glycol (20000)

[0139] 3mM ATP

[0140] 30mM creatine phosphate

[0141] 100ng / ul creatine kinase

[0142] 400ng / ul E. coli recA protein

[0143] 200ng / ul E. coli SSB protein

[0144] 60ng / ul E. coli recO protein

[0145] 40ng / ul E. coli recR protein

[0146] 60ng / ul E. coli recF protein

[0147] 8Units Bacillus subtilis DNA polymerase I

[0148] 50ng / ul Endonuclease IV

[0149] 450uM dNTPs

[0150] 250uM each upstream primer

[0151] 250uM each downstream primer

[0152] 120nM fluorescent probe

[0153] The amplification temperature was 4...

Embodiment 3

[0156] The design of all primers and probes gives priority to 8 common mycoplasmas (M.arginini, M.fermentans, M.hominis, M.orale, M.hyorhinis, M.salivarium, M.pirum and Acholeplamasalaidlaawii), and further Consider covering other mycoplasmas, and at the same time exclude the interference of E. coli, see the comparison results Figure 10 :

[0157] The probe and primer sequences are as follows:

[0158] Upstream primer sequence: 5'-CACCTCGATGTCGRCTCATCTCATCCTCGA-3' (Seq ID No 6), which can amplify all 23 kinds of mycoplasma;

[0159] The sequences of the three downstream primers are: 5'-CTAGGAACAGCTCTTCTCAATCTTCCTACGGG-3' (Seq ID No 7), which can amplify 6 species of mycoplasma including M.gallisepticum, M.pirum, M.pneumoniae, M.penetrans, M.capricolum, and Ureaplasma urealyticum ; 5'-GTAGCTCTCATCAATATTCCAACGCCCACAT-3'(SeqID No 8), can amplify M.bovis, M.bovoculi, M.fermentans, M.synoviae, M.pulmonis, M.flocculare, M.hyorhinis, M.hominis, M.arginini, M.arthritidis, M.hyosyn...

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Abstract

The invention provides a new detection method for detecting mycoplasma contamination in a cell culture process and a detection kit. According to the new detection method and the detection kit, relatively conservative rDNA in mycoplasma is taken as a detection target segment, the detection is performed with a recombinase and polymerase based isothermal amplification technology by combination of specific primers and probes, so that operation is simple and convenient, detection time is shortened, and specificity and accuracy of mycoplasma contamination detection are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting mycoplasma contamination through constant temperature amplification technology, and a combination of primers and probes used in the method. Background technique [0002] Cell culture technology is one of the essential core links in the fields of biopharmaceuticals, vaccine production and clinical cell therapy. However, a serious problem in the cultivation process is that the cells are often contaminated. The sources of contamination include mycoplasma, bacteria and fungi, among which mycoplasma is the most important source of contamination. [1-2] . After mycoplasma contamination, it can spread through serum, experimenters and contaminated cell culture, etc. [3-5] , leading to larger contamination of the cell sample. According to statistics, about 15%-35% of cell cultures in the world are contaminated by mycoplasma [6-7] . [0003] Mycoplasma is...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/35
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101
Inventor 张杨吴玉于继彬
Owner SUZHOU GENDX BIOTECH CO LTD
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