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Method for detecting and identifying mycoplasma

An identification method, mycoplasma technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high specificity, low cost, repeated steps, etc., and achieve the effect of rapid detection and reduced detection cost

Inactive Publication Date: 2005-10-05
武汉市第一医院
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Problems solved by technology

The PCR method is currently the most commonly used diagnostic method (references Kong, F., etc. 2001.Species-specific PCR for identification of common contaminated mollicutes in cell culture.Appl.Environ.Microbiol.67:3195-3200), but using PCR detection, The identification of multiple mycoplasmas has the disadvantages of time-consuming and repetitive steps
There is currently no rapid, sensitive, specific and low-cost method for the simultaneous detection of multiple mycoplasmas

Method used

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  • Method for detecting and identifying mycoplasma

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Embodiment

[0022] The instruments used include: -20°C and 4°C refrigerators, room temperature high-speed centrifuge (Eppendorf Company, Germany), PCR instrument (Eppendorf Company, Germany), Miniblotter (Immunetic Company, U.S.), hybridization box (Thermo Company, U.S.), cassette.

[0023] The reagents used are: Roche Respiratory Specimen DNA Extraction Kit (Roche Diagnostic Reagent Company, USA), Taq enzyme, dNTP, 10×PCR buffer, ultra-clean water, 0.5M NAHCO 3 (PH8.4), 0.1M NaOH, 16% w / v 1Ethyl-3-(3 Dimethylamino-propyl)carbodiimide (EDAC) (Sigma company, the United States), 20 × SSPE (0.2MNa 2 HPO 4 , 3.6M NaCl, 20mM EDTA, PH7.4), 2×SSPE / 0.1%SDS, 2×SSPE / 0.5%SDS, 2×SSPE, 1%SDS, 20mM EDTA, Biodyne C nylon membrane (Immunetic company, the United States), Streptavidin-peroxidase conjugate (Roche Diagnostics, USA), Enhanced chemiluminescence (ECL) developer (Amersham, UK), X-ray film (Amersham, UK).

[0024] Steps:

[0025] 1. Extract DNA using Roche Respiratory Specimen DNA Extraction ...

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Abstract

This invention is a MP testing and identifying method, including distill DNA of samples to be tested, use marked MP general lead to expand the DNA of pending tested samples in PCR method, hybridize the PCR expanded products with the MP species-specific probe markedly located on the nylon film, wash the un-hybridized PCR expanded products on the nylon film, display the hybridizing results and etc, and meanwhile, this invention also provides the MP general lead, the MP species-specific probe and the reagent boxes of testing and identifying MP. The probe of this invention is delicate, and the testing and identifying are fast sensitive and the cost is low.

Description

technical field [0001] The present invention relates to the detection and identification method of microorganisms, especially the detection and identification method of mycoplasma, specifically relates to the detection and identification of ten kinds of common cell contamination and pathogenic mycoplasma. Background technique [0002] Mycoplasma contamination is a common problem in cell culture experiments, the pharmaceutical industry, and the production of biologics, which can lead to experimental failure and decreased quality of biological products. Mycoplasma contamination has been reported in 15%-85% of cell cultures in various laboratories. Among the currently known more than 20 cell-contaminating mycoplasmas, Mycoplasma argininum (Mycoplasma arginin), Mycoplasma fermentans (Mycoplasma fermentans), Mycoplasma hyorhinis (Mycoplasma hyorhinis), Mycoplasma orale (Mycoplasma orale) and Mycoplasma reysterii (Acholeplasma laidlawii) is the most common. In cell culture, mor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王辉孔繁荣琳·吉尔伯特
Owner 武汉市第一医院
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