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Mycoplasma clearing reagent and application thereof

A technology of mycoplasma and reagent, applied in the field of mycoplasma removal reagent, can solve the problems of different, easy repeated infection of mycoplasma and the like

Inactive Publication Date: 2010-10-06
上海龙鼎医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, laboratory cell culture is different from clinical application, and in cell culture, mycoplasma is easy to be repeatedly infected. For important cell lines being tested, it is necessary to remove mycoplasma as soon as possible

Method used

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  • Mycoplasma clearing reagent and application thereof
  • Mycoplasma clearing reagent and application thereof
  • Mycoplasma clearing reagent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Configuration of mycoplasma removal reagent

[0050] Prepare 250ul reagent according to the following formula (each component is calculated by weight percentage):

[0051] Formulation 1: Gemifloxacin 1.4%

[0052] Minocycline 0.4%

[0053] Azithromycin 0.2%

[0054] Buffer 98%

[0055] Buffer formula:

[0056] NaOH 0.4%

[0057] Nacl 0.9%

[0058] Glucose 5%

[0059] H 2 O 93.7%

[0060] Formulation 2: Moxifloxacin 1.3%

[0061] Doxycycline 0.5%

[0062] Clarithromycin 0.1%

[0063] Buffer 98.1%

[0064] Buffer formula:

[0065] NaOH 0.2%

[0066] Nacl 1.0%

[0067] Glucose 8%

[0068] H 2 O 90.8%

[0069] Formula 3: Clinfloxacin 1.5%

[0070] Tigecycline 0.3%

[0071] Roxithromycin 0.3%

[0072] Buffer 97.9%

[0073] Buffer formula:

[0074] NaOH 0.6%

[0075] Nacl 0.9%

[0076] Glucose 5%

[0077] H 2 O 93.5%

[0078] Configuration method

[0079] 1. Buffer configuration

[0080] 1) Ultra-pure water 103.4kpa steam pressure temperature reaches 121.3℃, maintain 15-20 minutes moist heat steriliza...

Embodiment 2

[0086] Example 2 Test of mycoplasma removal effect of mycoplasma removal reagent

[0087] The mycoplasma scavenging reagent prepared by formula 1 in Example 1 was used as follows figure 1 Said includes the following steps:

[0088] 1. Resuscitate the cells, plant the plate after adjustment, and plant 1x10 in a 6cm plate 6 Cells

[0089] 2. After the cells adhere to the wall the next day, dilute the mycoplasma removal reagent into a medium containing 5% FBS without dual antibody at a ratio of 2000x, and use this medium for culture during subsequent passages;

[0090] 3. After acting for 7 days, pay attention to observe and record the cell status during this process, and use the medium containing mycoplasma scavenging reagent to maintain when changing the medium or passage;

[0091] After 4.7 days, change to normal complete medium without antibiotics and culture for 2 days, and increase the volume of each plate to 4-5ml;

[0092] After 5.2 days, aspirate 500ul of culture supernatant, and p...

Embodiment 3

[0096] Example 3 Mycoplasma scavenging effect test of reagent

[0097] experiment equipment:

[0098] Ultrasound stage, water bath, centrifuge, negative 80 degree refrigerator, PCR machine, electrophoresis tank, gel imager, electrophoresis,

[0099] Sterile PCR tube Sterile 1.5ml EP tube PCR tube rack Sterile pipette tip Cotton ball

[0100] Pipette (1ml 200ul 10ul 2ul)

[0101] Experimental reagents:

[0102] Sterile ultrapure water Sample to be tested 75% sterile alcohol Mycoplasma spray (prepared in Example 1)

[0103] Germ-free test medium

[0104] Nested PCR Mycoplasma Rapid Detection Kit (Shanghai Jisheng Pharmaceutical Company)

[0105] 2% agarose gel EB electrophoresis buffer Mark (DL250+) 6x Loading Buffer

[0106] Experimental steps:

[0107] Refer to the method of Example 2 to use a mycoplasma scavenging reagent.

[0108] Use the following method to use the nested PCR Mycoplasma rapid detection kit:

[0109] 1. Collect the samples to be tested and store in the refrigerator at minus 8...

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Abstract

The invention relates to a mycoplasma clearing reagent and application thereof. The mycoplasma clearing reagent of the invention comprises the following components in percentage by weight: 1.3 to 1.5 percent of fluoroquinolones antibiotics, 0.3 to 0.5 percent of tetracyclines derivatives, 0.1 to 0.3 percent of macrolides antibiotics and 97.7 to 98.3 percent of buffer solution, can most effectively inhibit and kill mycoplasmas, and has a short period, namely, all the mycoplasmas can be killed in only one week without influencing the metabolism of cells per se; and the treated cells have smooth surfaces, substantially have no black particles and are difficult to be re-infected by the mycoplasmas.

Description

Technical field [0001] The invention relates to a reagent for removing mycoplasma and its application. Background technique [0002] Mycoplasma is the most common pollutant in cell culture that is not easy to be detected and interferes with experimental results. About 30.3% to 50.5% of cell cultures are polluted by mycoplasma. The sources of mycoplasma contamination in cultured cells include: some products of animal origin used, such as serum or cell lines that have been contaminated with mycoplasma. The concentration of mycoplasma in the cell culture supernatant can reach 107 cells / ml, and the cultured cells look normal. Observed by the naked eye or under an ordinary optical microscope, the contaminated cells have no obvious changes. Due to competition for nutrition and mycoplasma's toxic metabolites, mycoplasma contamination can significantly affect the physiological activity of cultured cells, greatly reducing the authenticity and reliability of research results. [0003] Most...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
Inventor 王海峰韦有恒曹跃琼高博
Owner 上海龙鼎医药科技有限公司
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