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Method for concentrating sample constituents and amplifying nucleic acids

A nucleic acid and biological sample technology, applied in biochemical equipment and methods, analytical materials, recombinant DNA technology, etc., can solve problems such as difficulty in the preparation of μ-TAS, achieve time and cost savings, and reduce the number of effects

Inactive Publication Date: 2012-05-16
ROBERT BOSCH GMBH
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  • Abstract
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Problems solved by technology

[0008] However, not only is the production of robust μ-TAS for diagnostics difficult due to the many working steps between sample handling and PCR reaction, but also due to the fact that, for example, for the analysis of cell- or virus-containing More than the amount of just a few drops used for analysis, since only very small numbers of cells or viruses are often present in this medium

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  • Method for concentrating sample constituents and amplifying nucleic acids
  • Method for concentrating sample constituents and amplifying nucleic acids
  • Method for concentrating sample constituents and amplifying nucleic acids

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Embodiment Construction

[0076] figure 1 A schematic flow diagram of the method of the invention using the separation system is shown.

[0077] A biological sample such as urine (which contains germs such as E. coli) is applied to the filter. The nucleic acid to be amplified is DNA, and the amplification is performed by means of PCR.

[0078] In step 1, each ml contains 10 4 -10 7 A macroscopic sample volume (10 ml) of the bacterial suspension 102 of 1 bacteria is passed through the filter (here the silica fiber mat 101 ) at a pressure of maximum 0.5 bar for 10 minutes (ie 1 ml / min). E. coli is retained on the silica fiber mat with a separation of 95%-99%, so that the suspension 103 then contains almost no bacteria 102. Then in step B, wash the filter 101 with 100-500 μl of PCR buffer to create optimal conditions for PCR. In step C, the PCR solution, ie 30 μl PCR master mix containing dNTPs, buffer, Taq-polymerase, primers and PEG, is added and the PCR cycle is started. In the PCR reaction, the...

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Abstract

A method is disclosed for concentrating sample constituents and for multiplying nucleic acids from a biological sample which are containing in the sample constituents. The nucleic acids are amplified on the same filter on which the sample constituents are also separated off.

Description

technical field [0001] The present invention relates to a method for concentrating sample components and amplifying nucleic acids contained in the sample components. Background technique [0002] In the diagnosis of infectious diseases, the demonstration of antibiotic resistance is of increasing importance due to the continuous spread of antibiotic resistance such as MRSA and fluoroquinolone resistance against Escherichia coli and bacteria causing tuberculosis, sepsis or pneumonia. [0003] In routine diagnosis, these resistances are mostly learned on the basis of culture. But this cell culture-based resistance assay is a time-consuming method, as it takes 2-3 days for cultivation and evaluation. Resistance can be determined remarkably quickly by means of bacterial DNA analysis. To do this, the bacterial DNA is amplified. Such amplification is possible, for example, by the concerted action of DNA and RNA polymerases (nucleic acid sequence-based amplification; NASBA), by p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12M1/00
CPCG01N33/5302C12Q1/6806B01L2200/10C12Q2523/303C12Q2565/137C12N15/1017
Inventor P.罗塔歇尔J.魏勒S.明希M.道布
Owner ROBERT BOSCH GMBH
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