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Human umbilical cord and placenta protection solution mycoplasma detection primer pair, kit and detection method thereof

A technology for detection kits and protective solutions, which is applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc. It can solve problems such as inaccurate experimental results, unfavorable stem cell culture, and long culture time. Achieve high sensitivity, prevent mycoplasma from entering the cell culture room, and take a short time

Inactive Publication Date: 2017-07-21
YUNNAN SHUNXI REGENERATION MEDICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The culture method takes a long time, a detection cycle is 28 days, and the sensitivity is poor; the DNA fluorescence staining method makes the experimental results inaccurate due to the existence of background fluorescence
However, other methods are not conducive to the cultivation of stem cells due to the limitation of price and experimental conditions, or because of low sensitivity, complicated operation, and high cost.

Method used

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  • Human umbilical cord and placenta protection solution mycoplasma detection primer pair, kit and detection method thereof
  • Human umbilical cord and placenta protection solution mycoplasma detection primer pair, kit and detection method thereof
  • Human umbilical cord and placenta protection solution mycoplasma detection primer pair, kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of Human Umbilical Cord and Placenta Protection Liquid Mycoplasma Detection Kit

[0045] 1. Primer synthesis

[0046] The following primer pairs were artificially synthesized:

[0047] Upstream primer Myco-F: 5'-ggcgaatgggtgagtaacacg-3'; (SEQ ID NO.1)

[0048] Downstream primer Myco-R: 5'-cggataacgcttgcgacctatg-3'. (SEQ ID NO.2)

[0049] Primers were prepared with ultrapure water and diluted to 2.5 μM, and stored at -20°C for later use.

[0050] 2. Preparation of positive control

[0051] In the process of stem cell culture, the cells that are confirmed to be contaminated by mycoplasma by the separation and culture method, use a Pasteur pipette or a pipette to absorb 1 mL of the cell culture supernatant, and take 3 μL as the sample to be tested, and then amplify by PCR using the primer pair in the above step After that, it was analyzed by agarose gel electrophoresis with a mass percentage of 2%, and a clear band was obtained at 400-500 bp, whi...

Embodiment 2

[0058] Example 2 Application of Human Umbilical Cord and Placenta Protection Liquid Mycoplasma Detection Kit

[0059] Samples were tested using the kit prepared in Example 1.

[0060] 1. Obtaining samples to be tested

[0061] According to the aforementioned method, take three tubes of the umbilical cord protection solution not polluted by mycoplasma as the test samples of group A; three tubes of the umbilical cord protection solution polluted by mycoplasma as the test samples of group B; Three tubes were used as test samples of group C; three tubes of the placenta protection solution contaminated by mycoplasma were used as test samples of group D.

[0062] 2. PCR amplification

[0063] 1. Use groups A, B, C, and D as samples to be tested, and add the following reagents into the PCR reaction tube according to the amplification system:

[0064] 2×power Taq PCR MasterMix 5μL;

[0065] Upstream primer 2.5μM 1μL (2.5μM);

[0066] Downstream primer 2.5μM 1μL (2.5μM);

[0067]...

Embodiment 3

[0072] Example 3 Sensitivity Detection of Human Umbilical Cord and Placenta Protection Liquid Mycoplasma Detection Kit

[0073] Samples were tested using the kit prepared in Example 1.

[0074] 1. Obtaining samples to be tested

[0075] In the process of stem cell culture, the cells that are confirmed to be contaminated by mycoplasma by the separation and culture method, use a Pasteur pipette or a pipette to absorb 1 mL of the cell culture supernatant, and take 3 μL as the sample to be tested, and then amplify by PCR using the primer pair in the above step After that, it was analyzed by agarose gel electrophoresis with a mass percentage of 2%, and a clear band was obtained at 400-500bp, and the product was recovered by cutting the gel.

[0076] The PCR reaction system is PCR reagent 2×power Taq PCR MasterMix 5 μL, upstream primer 2.5 μM 1 μL, downstream primer 2.5 μM 1 μL, sample to be tested 3 μL;

[0077] The PCR amplification program is: 94° C. for 3 min; 94° C. for 30 s,...

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Abstract

The invention relates to a human umbilical cord and placenta protection solution mycoplasma detection primer pair, a kit and a detection method thereof, and belongs to the technical field of in-vitro diagnosis reagents. The kit comprises a primer pair, a PCR (Polymerase Chain Reaction) reagent, a positive control, a negative control and a PCR reaction tube, wherein the primer pair comprises an upstream primer Myco-F: 5'-ggcgaatgggtgagtaacacg-3', and a downstream primer Myco-R: 5'-cggataacgcttgcgacctatg-3'. The detection kit provided by the invention is simple and convenient to operate, short in time and very high in sensitivity, whether cells are contaminated by mycoplasma or not can be detected before culture, the economic loss caused by aimless cell culture can be avoided, the mycoplasma can be fundamentally prevented from entering a cell culture chamber, and possibility of mycoplasma cross contamination of cells can be reduced.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnostic reagents, in particular to a primer pair for detecting mycoplasma in human umbilical cord and placenta protection solution, a kit and a detection method thereof. Background technique [0002] Mycoplasma ( mycoplasma ), also known as mycoplasma, is one of the smallest microorganisms that can survive independently between bacteria and viruses. It is a type of prokaryotic microorganisms that lack cell walls. μm, can pass through the bacteria filter (0.22μm), and is widely distributed in nature. In the initial stage of mycoplasma contamination in the cell culture process, the cells often do not cause obvious lesions, nor do they cause the culture medium to be turbid, which is difficult to detect with the naked eye, and there is no obvious change in the cells observed under a high-power microscope. In the late stage of pollution, mycoplasma adsorbs on the cell surface, destroys the integr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2545/113
Inventor 张瑞丽李一佳汪鉴
Owner YUNNAN SHUNXI REGENERATION MEDICAL ENG CO LTD
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