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Method for detecting escherichia coli pathogenic bacteria from clinical blood

A technology of Escherichia coli and pathogenic bacteria, applied in the field of detection of pathogenic bacteria of blood infection, can solve the problems of untimely treatment of infected patients, tense relationship between doctors and patients, etc., and achieve the effect of improving efficiency and sensitivity, improving detection efficiency, and simple and convenient operation

Inactive Publication Date: 2019-11-26
卓源健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, for the detection of pathogenic microorganisms in clinical blood, the traditional inspection method in the hospital still needs to go through complex and time-consuming intermediate steps such as cultivation. Escherichia coli, which is easy to cultivate, also needs to be cultured overnight by smearing and streaking. It usually takes about 3-5 days for subsequent physiological and biochemical or drug resistance analysis
This will lead to a series of problems such as untimely treatment of infected patients, and what's more, it will directly lead to a tense relationship between doctors and patients.

Method used

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  • Method for detecting escherichia coli pathogenic bacteria from clinical blood
  • Method for detecting escherichia coli pathogenic bacteria from clinical blood
  • Method for detecting escherichia coli pathogenic bacteria from clinical blood

Examples

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Effect test

Embodiment 1

[0039] Example 1: Design of specific PCR primers

[0040] A specific PCR primer is designed by using the genome DNA sequence of Escherichia coli, and the specific PCR primer includes a forward primer and a reverse primer. The forward primer is shown in SEQ ID NO.1: 5'-TAATGTTCTGCGACGCTCAC-3'. The reverse primer is shown in SEQ ID NO.2: 5'-CCCGGCTAACGTATCCAC-3'. The length of the amplified fragment of the primers is 200bp.

Embodiment 2

[0042] Escherichia coli specific primers were used to detect pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Listeria monocytogenes and Klebsiella pneumoniae added to the blood. In this example, OD is added to every 100 μL of blood 6000.1 of the above-mentioned various pathogenic bacteria. This example includes the extraction and PCR amplification detection of bacterial genomic DNA in blood. This method comprises the following steps:

[0043] (1) Collect 100 μL cultured OD 600 =0.1 for Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Listeria monocytogenes and Klebsiella pneumoniae. Centrifuge at 12000 rpm for 1 minute to collect the precipitated bacteria.

[0044] (2) Take 100 μL of fresh blood from 6 groups, and add Escherichia coli, Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa precipitated in step (1) into the 6 groups of 100 μL fresh blood , Listeria monocytogenes and...

Embodiment 3

[0059] Detection of Escherichia coli added in blood by using Escherichia coli specific primers. In this example, 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 ,

[0060] 10 2 , 10 1 , 10 0 CFU of Escherichia coli. This example includes the extraction and PCR amplification detection of bacterial genomic DNA in blood. This method comprises the following steps:

[0061] (1) Collect 10 cultivated 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 CFU of Escherichia coli. Centrifuge at 12000 rpm for 1 minute to collect the precipitated bacteria.

[0062] (2) Take 100 μL of fresh blood from 9 groups, and add Escherichia coli cells with different numbers of colonies precipitated in step (1) to the 100 μL fresh blood of 9 groups, resuspend the precipitated cells, and mix well by pipetting.

[0063] (3) Add 100 μL of enzyme lysate to it, and incubate at 37° C. for 15 minutes. The enzyme lysate contains: 0.1mg / mL ribonuclease A, 1mg / mL proteinase K, 0.05mg / mL bro...

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Abstract

The present invention provides a method for detecting escherichia coli pathogenic bacteria from clinical blood. The method comprise the following steps: S1, escherichia coli genome DNA in blood is extracted; and S2, PCR detection of escherichia coli in blood is detected: S2.1, a DNA sequence of the escherichia coli genome is used to design specific PCR primers, forward primers are shown in SEQ IDNO.1 and reverse primers are shown in SEQ ID NO.2; and an amplified fragment length of the primers is 200 bp; S2.2, the escherichia coli genome DNA is used as a template and the specific PCR primers are used for PCR amplification; and S2.3, agarose gel electrophoresis and detection are conducted on PCR amplification products to amplify a fragment of 200 bp in length to determine detection of escherichia coli. A detection limit of the escherichia coli in the blood reaches 100 CFU / mL, rapid extraction of the escherichia coli genome DNA in the clinical blood is directly conducted and rapid PCR detection of specific genes is conducted, which can realize timely and effective diagnosis of escherichia coli infection in the blood.

Description

technical field [0001] The invention relates to the technical field of detecting blood infection pathogenic bacteria, in particular to a method for detecting Escherichia coli pathogenic bacteria from clinical blood. Background technique [0002] Escherichia coli, commonly known as E. coli, is a type of bacteria that is commonly found in the human body. General Escherichia coli is not pathogenic, but other species such as diarrhea-causing Escherichia coli can cause human diseases, and childhood sepsis is also closely related to pathogenic Escherichia coli. Bloodstream infections caused by Escherichia coli have also emerged as culprits in bloodstream infections caused by Gram-negative bacteria. [0003] At present, for the detection of pathogenic microorganisms in clinical blood, the traditional inspection method in the hospital still needs to go through complex and time-consuming intermediate steps such as cultivation. Escherichia coli, which is easy to cultivate, also needs...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12R1/19
CPCC12Q1/689C12Q1/686G01N2333/245C12Q2565/125
Inventor 李敬王旭孙宝林郭建吴谨
Owner 卓源健康科技有限公司
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