Method for extracting methicillin-resistant staphylococcus aureus DNA (Deoxyribonucleic Acid)

A methicillin, staphylococcus-resistant technology, applied in DNA preparation, recombinant DNA technology and other directions, can solve the problems of complex process, limited sensitivity, containing harmful chemicals, etc., and achieve the effect of simple preparation process and high detection sensitivity

Inactive Publication Date: 2013-06-19
SHANGHAI PUTUO DISTRICT PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are commercial kits at home and abroad that can extract bacterial DNA for subsequent PCR reactions, but the sensitivity for detecting Staphylococcus aureus is 10 5 -10 6 CFU, Portion Only 10 7 -10 8 CFU, and the extraction steps are complicated, involving multiple centrifugation, supernatant passing through the column, washing and other steps
There are also reports in the literature that some methods for extracting bacterial genomic DNA require extraction steps such as

Method used

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  • Method for extracting methicillin-resistant staphylococcus aureus DNA (Deoxyribonucleic Acid)
  • Method for extracting methicillin-resistant staphylococcus aureus DNA (Deoxyribonucleic Acid)
  • Method for extracting methicillin-resistant staphylococcus aureus DNA (Deoxyribonucleic Acid)

Examples

Experimental program
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Example Embodiment

[0025] Example 1

[0026] 1) Configuration of lysate:

[0027] Weigh Tris (tris(hydroxymethylaminomethane, molecular weight 121.14)), Na2-EDTA (ethylenediaminetetraacetic acid sodium salt, molecular weight 336.21), chelex100 (weak cation chelating resin), lysozyme, lysozyme (lysostaphin) and proteinase K are dissolved in an appropriate amount of sterile water, then an appropriate volume of Triton x-100 solution is added, the pH value is adjusted to 8.0 with HCL (or NaOH), and the volume is made constant with sterile water.

[0028] The final concentration of the solution is as follows (pH8.0): 20mM (ie mmol / L) Tris (tris(hydroxymethylaminomethane)); 2 mM Na2-EDTA (ethylenediaminetetraacetic acid sodium salt); 1.0%(v / v) Triton x-100; 20mg / ml lysozyme; 0.10g / ml chelex100 (weak cation chelating resin); 100ug / ml lysostaphin; 2mg / ml proteinase K; the rest is sterile water;

[0029] 2) 5 clinically isolated Staphylococcus aureus strains, including 1 strain of MSSA and 4 strains of MRSA, we...

Example Embodiment

[0031] Example 2

[0032] The configuration of the lysis solution in this embodiment is as follows:

[0033] Weigh Tris (tris(hydroxymethylaminomethane, molecular weight 121.14)), Na2-EDTA (ethylenediaminetetraacetic acid sodium salt, molecular weight 336.21), chelex100 (weak cation chelating resin), lysozyme, lysozyme (lysostaphin) and proteinase K are dissolved in an appropriate amount of sterile water, then an appropriate volume of Triton x-100 solution is added, the pH value is adjusted to 8.0 with HCL (or NaOH), and the volume is made constant with sterile water.

[0034] The final concentration of the solution is as follows (pH8.0): 20mM (ie mmol / L) Tris (tris(hydroxymethylaminomethane)); 2 mM Na2-EDTA (ethylenediaminetetraacetic acid sodium salt); 1.0%(v / v) Triton x-100; 20mg / ml lysozyme; 0.05g / ml chelex100 (weak cation chelating resin); 90ug / ml lysostaphin; 2mg / ml proteinase K; the rest is sterile water.

[0035] Then take 5 strains of clinically isolated Staphylococcus aureu...

Example Embodiment

[0036] Example 3

[0037] The configuration of the lysis solution in this embodiment is as follows:

[0038] Weigh Tris (tris(hydroxymethylaminomethane, molecular weight 121.14)), Na2-EDTA (ethylenediaminetetraacetic acid sodium salt, molecular weight 336.21), chelex100 (weak cation chelating resin), lysozyme, lysozyme (lysostaphin) and proteinase K are dissolved in an appropriate amount of sterile water, then an appropriate volume of Triton x-100 solution is added, the pH value is adjusted to 8.0 with HCL (or NaOH), and the volume is made constant with sterile water.

[0039] The final concentration of the solution is as follows (pH 8.0): 20mM (ie mmol / L) Tris (tris(hydroxymethylaminomethane)); 2 mM Na2-EDTA (ethylenediaminetetraacetic acid sodium salt); 1.0%(v / v) Triton x-100; 20mg / ml lysozyme; 0.15g / ml chelex100 (weak cation chelating resin); 100ug / ml lysostaphin; 1.8mg / ml proteinase K; the rest is sterile water .

[0040] Then take 5 strains of clinically isolated Staphylococcus...

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Abstract

The invention provides a method for extracting methicillin-resistant staphylococcus aureus DNA (Deoxyribonucleic Acid). The method comprises the following steps of: preparing a lysis solution (pH 8.2) by the following components in final concentration: 20mM of Tris, 2mM of Na2-EDTA (Ethylene Diamine Tetraacetic Acid), 1.0% of Tritonx-100, 20mg/ml of lysozyme, 0.05g/ml-0.15g/ml of chelex100, 90mu g/ml-100mu g/ml of lysostaphin, 1.8mg/ml-2mg/ml of protease K and the balance of sterile water; getting and suspending 5-10 staphylococcus aureus bacterial colonies, which grow on the solid culture medium, in the 100mu l of the lysis solution, centrifuging for 30 minutes at 56 DEG C, 10 minutes at 100 DEG C and 10 minutes at the speed of 12000rpm; and getting and using the liquid supernatant for the PCR (Polymerase Chain Reaction).

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a method for extracting methicillin-resistant Staphylococcus aureus DNA. Background technique [0002] Staphylococcus aureus is an important pathogen, and the isolation rate of methicillin-resistant Staphylococcus aureus (MRSA) in Staphylococcus aureus has exceeded 50%. Treatment costs have increased significantly. The rapid diagnosis of MRSA can be carried out within a few hours with molecular biological methods, but the basis of its work is the extraction of MRSA genomic DNA. Staphylococcus aureus is a Gram-positive bacterium with a thicker cell wall (20-80nm) and 5-15 layers of peptidoglycan, while the cell wall of Gram-negative bacteria is only 8-11nm with only 1-2 layers of peptidoglycan . Incomplete bacterial lysis, DNA not released, or polymerase chain reaction (PCR) inhibitors in the lysed sample can lead to false negative results, significantly reducing a...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 金姝朱晴晖邹玉涵黄德魁童平
Owner SHANGHAI PUTUO DISTRICT PEOPLES HOSPITAL
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