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Preparation method of pathogenic bacterium DNAs (Deoxyribonucleic Acids) in clinical blood sample and kit

A technology of blood samples and pathogenic bacteria, applied in the field of molecular diagnostics of pathogenic bacterial infection, can solve the problems of PCR reaction difficulties and achieve the effect of improving specificity and sensitivity and avoiding interference

Inactive Publication Date: 2011-11-23
GUANGZHOU INNOGENIX BIOMEDICINE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The presence of the vast majority of non-pathogenic bacterial DNA in clinical samples has brought serious problems and difficulties to the application of PCR reactions in the diagnosis of infectious diseases

Method used

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  • Preparation method of pathogenic bacterium DNAs (Deoxyribonucleic Acids) in clinical blood sample and kit
  • Preparation method of pathogenic bacterium DNAs (Deoxyribonucleic Acids) in clinical blood sample and kit
  • Preparation method of pathogenic bacterium DNAs (Deoxyribonucleic Acids) in clinical blood sample and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Lysis of blood cells by ox gall powder

[0031] The ox gall powder (produced by sigma company, article number is Fluka, B3883) aqueous solution is that a certain amount of ox gall powder is dissolved in water, and the dissolution is complete.

[0032] Take 1 milliliter of fresh human blood, mix it with 4 milliliters of ox gall powder aqueous solutions with concentrations of 0, 15, 20, 25, 30 and 50 g / L respectively, and place it at room temperature (23-25° C.). Take 10 microliters of samples at regular intervals, put them into 200 microliters of normal saline, and observe the degree of blood cell lysis with a microscope.

[0033] The results are shown in Table 1. As can be seen from Table 1, ox gall powder can rapidly and completely lyse blood cells. In detail, even if the effect is as long as 5 hours, blood cells will not be lysed in normal saline (no ox gall powder), 12g / L ox gall powder can only partially lyse blood cells, and ox gall powder with a concentration abo...

Embodiment 2

[0037] 1. Preparation of simulated clinical blood samples

[0038] A typhoid colony was inoculated into 2 ml of tryptone soybean broth (OXOID Tryptone Soya Broth, UK: TSB for short), and cultured on a constant temperature shaker at 37° C. at 200 rpm for 2 hours to obtain a typhoid culture medium. First dilute the Salmonella typhi culture solution to A 600 Next, the OD was 0.25, and then a further 10-fold serial dilution was made. Take 100 microliters from each of the dilutions and culture them on tryptone soybean agar medium at 37°C to determine the content of Salmonella typhi in each dilution.

[0039] Human blood is drawn with a sterile syringe and immediately placed into a test tube containing heparin. 900 ml of fresh blood was mixed with 100 microliters of dilutions containing different numbers of typhoid bacteria. The mixture thereof is a simulated clinical blood sample, and the simulated clinical blood sample is used for the separation and preparation of DNA.

[0040...

Embodiment 3

[0053] Effects of different concentrations of ox gall powder aqueous solution on the biological activity of Micrococcus deoxyribonuclease

[0054] (1) Get 7 20 microliters containing 0.5 micrograms of human blood DNA, and respectively containing 0, 10, 30, 50, 70 and 90 g / L of ox gall powder aqueous solution, and put them into reaction tubes. Add 2x10 to each 3 1 unit of Micrococcal DNase, mixed well and left at room temperature for 10 minutes, and then checked for DNA degradation in the reaction solution by agarose gel electrophoresis. The result is as image 3 Shown: micrococcal deoxyribonuclease can completely degrade human blood DNA, and under the reaction conditions of ox gall powder concentration up to 90g / L, micrococcal deoxyribonuclease still has its biological activity.

[0055] (2) Take 10 microliters of ox gall powder aqueous solutions with a concentration of 0, 50 or 100 g / L, and put them into a reaction tube. Add 10 microliters of micrococcal deoxyribonuclease ...

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Abstract

The invention discloses a preparation method of pathogenic bacterium DNAs (Deoxyribonucleic Acids) in a clinical blood sample and a kit. The method comprises the following steps of: mixing the clinical blood sample with ox-bile or ox-bile powder and making the ox-bile or ox-bile powder crack non-pathogenic bacterium cells; during or after cracking of the non-pathogenic bacterium cells, adding desoxyribonuclease for degrading DNAs released after cracking of the non-pathogenic bacterium cells; centrifuging the mixture obtained in the previous step and collecting precipitates; and preparing pathogenic bacterium DNAs from the precipitates with the conventional method. In the invention, the ox-bile or ox-bile powder is used for cracking the non-pathogenic bacterium cells in the clinical blood sample to release the non-pathogenic bacterium cell DNAs according to the characteristic of the tolerance of malignant bacteria and fungi in the clinical sample to the ox-bile and the ox-bile powder, and desoxyribonuclease is used for degrading to selectively remove non-pathogenic bacterium DNAs, so that the pathogenic bacterium DNAs are enriched. By undergoing a PCR (Polymerase Chain Reaction) on the DNAs, the interference of most of non-pathogenic bacterium DNAs on the PCR detection of pathogenic bacteria can be avoided, and the specificity and sensitivity of the PCR can be enhanced.

Description

Technical field: [0001] The invention belongs to the field of molecular diagnosis of pathogenic bacteria infection, and in particular relates to a preparation method for enriching and extracting pathogenic bacteria DNA from clinical blood samples, and a kit used in the method. Background technique: [0002] The current standard method used in clinical microbiology laboratories to identify systemic infections in patients is blood culture. However, the sensitivity of blood culture methods is greatly affected by the use of antibiotics and the number of pathogenic bacteria contained in the sample. It is even impossible for some pathogenic bacteria to grow in vitro. In addition, the blood culture method has a long period, and it usually takes 2 to 5 days to get the result, which greatly delays the effective treatment of the patient. [0003] In recent years, nucleic acid polymerase chain reaction (PCR) has gradually been widely used in the field of molecular diagnosis of infect...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12Q1/04
Inventor 周立庆
Owner GUANGZHOU INNOGENIX BIOMEDICINE TECH
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