PCR (Polymerase Chain Reaction) kit for detecting mycoplasma pollution of cell culture and use method of PCR kit

A cell culture and mycoplasma technology, applied in the field of microbial detection, can solve the problems of expensive equipment and high detection cost, and achieve the effects of accurate and reliable detection, high sensitivity and fast speed

Pending Publication Date: 2022-07-15
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is straightforward and simple, its disadvantages are high detection cost and expensive equipment

Method used

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  • PCR (Polymerase Chain Reaction) kit for detecting mycoplasma pollution of cell culture and use method of PCR kit
  • PCR (Polymerase Chain Reaction) kit for detecting mycoplasma pollution of cell culture and use method of PCR kit
  • PCR (Polymerase Chain Reaction) kit for detecting mycoplasma pollution of cell culture and use method of PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Kit: dH2O, upstream primer, lyophilized powder of downstream primer, Taq enzyme, PCRBuffer.

[0027] Wherein, the upstream and downstream primer sequences are respectively:

[0028] For: 5'-CCAGCAAGCCGCGGTAATACATAGG-3'

[0029] Rev: 5'-GTGGACTACYAGGGTATCTAATCC-3'

[0030] The quantity of the PCRBuffer is 10 tubes / box.

[0031] The specific methods of using the above kits are as follows:

[0032] A. Pre-denaturation: perform pre-denaturation at a temperature of 95°C for 3 minutes;

[0033] B. Denaturation: keep the temperature unchanged for 30s;

[0034] C. Annealing: the temperature drops to 55℃, and the time is 30s;

[0035] D. Extension: maintained at 72°C for 35s;

[0036] Steps A-D were cycled 35 times, and finally maintained at 72 °C for 10 min, and the temperature was lowered to 4 °C.

[0037] This method was used to detect PCR products of serially diluted cell cultures when genomic DNA was diluted up to 10 6 When the concentration is 0.003ng / μl, the speci...

Embodiment 2

[0040] Four cell cultures of A: SLA-1, B: ST2, C: SLA-3, D: marc145 were detected using the kit described in Example 1. After 1.0% agarose gel electrophoresis, the PCR amplification products of the three cell cultures A, B, and C had specific amplification bands at the size of 280 bp ( Figure 4 ), consistent with the expected size of the mycoplasma bands, indicating that there may be mycoplasma contamination during the culture. However, there were no bands in the electrophoresis results of the PCR products of the D cell culture, indicating that they were not contaminated by mycoplasma.

Embodiment 3

[0042] Commercially available DMEM medium, fetal bovine serum, DMEM medium containing fetal bovine serum and PBS were detected using the same PCR system and method as in Example 2. like Figure 5 As shown, the results of 5 (positive control) and 6 (negative control) were normal results, but no specific mycoplasma bands were amplified in 1, 2, 3, and 4. This suggests that mycoplasma contamination did not originate from these materials.

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Abstract

The invention belongs to the technical field of microbiological detection, and discloses a PCR (Polymerase Chain Reaction) kit for detecting mycoplasma pollution of a cell culture and a use method of the PCR kit. DH2O, upstream primer freeze-dried powder, downstream primer freeze-dried powder, Taq enzyme and PCR (Polymerase Chain Reaction) Buffer are arranged in the kit, the sequences of the upstream primer and the downstream primer are respectively Forr: 5 '-CCAGCAAGCCGGTAATACATAGG-3' Rev5: 5 '-GTGGACTACYAGGGTATCTAATCC-3', and the specific use method of the kit comprises the following steps: pre-denaturation, denaturation, annealing, extension and circulation for 35 times. According to the invention, PCR detection of the mycoplasma is utilized, DNA replication in vivo is simulated in vitro, renaturation and denaturation of DNA are controlled through temperature change, the primer is used as a promoter, and a specific nucleic acid sequence of the mycoplasma can be amplified under the action of Taq enzyme and dNTP, so that the primer can be used for identifying various mycoplasmas.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a PCR kit for detecting mycoplasma contamination in cell cultures and a method for using the same. Background technique [0002] Most of the mycoplasmas are mainly spherical, with a three-layered cell membrane with great variability, which is pathogenic to humans and animals and can grow and reproduce on inanimate artificial media. The genome of Mycoplasma is mostly double-stranded DNA interspersed throughout the cell in nucleoid or non-formed nuclear regions. The cell membrane of mycoplasma contains sterols, which play a great role in maintaining the integrity of the cell membrane. Gram staining is not easy to stain, and various characteristics of mycoplasma are enough to show that it is not easy to be detected and removed. [0003] When cells are contaminated with Mycoplasma, the expression of DNA, RNA and proteins in cells will change to varying de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12R1/35
CPCC12Q1/689C12Q1/686C12Q2565/125
Inventor 张正韬熊可曹钧雄肖安超晏彦瑾康静怡王凯悦王钦富
Owner DALIAN UNIV
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