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Method for efficiently removing host nucleic acid in blood flow infection sample

A technique of in-sample and blood flow, applied in the fields of biochemical equipment and methods, determination/examination of microorganisms, etc.

Pending Publication Date: 2021-05-14
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the reports on PMA are mainly used to study the activity of bacteria, and there is no report on the use of PMA to remove host nucleic acids.

Method used

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  • Method for efficiently removing host nucleic acid in blood flow infection sample
  • Method for efficiently removing host nucleic acid in blood flow infection sample
  • Method for efficiently removing host nucleic acid in blood flow infection sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example microorganisms used in bloodstream infection in Example 1, Comparative Example 1, Example 2, Example 3 and Example 4 are Escherichia coli, which are recorded in the following documents: Zhao Chengna, Zhang Chao, Zheng Yuling, Liu Peng, Jiang Yongqiang, Establishment and evaluation of PCR array method for detection of 6 common childhood suppurative meningitis pathogens, Military Medicine, Volume 41, Issue 11, November 2017. The exemplary microorganism used in bloodstream infection in Example 5 is Staphylococcus aureus, which is recorded in the following documents: Li Qian, Liu Peng, Zhang Yan, Zhang Chao, Huang Wenhua, Zheng Yuling, Wang Hongquan, Jiang Yongqiang, Detecting 4 kinds of bloodstream infections Evaluation of the combination of targeted sequence enrichment and sequencing of pathogens, Military Medicine, Volume 42, Issue 1, January 2018.

[0098] Saponin: Sigma-Aldrich, Cat. No. S7900-100G; CAS No. 8047-15-2, EINECS No. 232-462-6; https: / / www.sigmaaldr...

Embodiment 2

[0150] The optimization of embodiment 2, PMA concentration

[0151] 1. Preparation of mock samples

[0152] Escherichia coli was resuspended in PBS buffer to obtain a bacterial concentration of 10 8 -10 9 CFU / ml Escherichia coli broth.

[0153] Each mock sample is 200 μl. The preparation method of each simulated sample: take 180 μl of healthy human whole blood and 20 μl of Escherichia coli bacteria liquid, mix well, and obtain simulated samples. Prepare multiple mock samples.

[0154] 2. Pre-processing

[0155] Basically the same as step 2 of embodiment 1.

[0156] The only difference is that: in step 3, 100 μl of PMA solutions with different concentrations were added; an equal volume of sterile water was used to replace the PMA solution as a 0-concentration PMA solution.

[0157] 3. DNA extraction

[0158] Take the pre-treated sample obtained in step 2, and use The IndiSpin Pathogen Kit to extract DNA to obtain a DNA sample.

[0159] Take 200 μl of simulated sample (...

Embodiment 3

[0163] Embodiment 3, optimization of photolysis time

[0164] 1. Preparation of mock samples

[0165] Escherichia coli was resuspended in PBS buffer to obtain a bacterial concentration of 10 8 -10 9 CFU / ml Escherichia coli broth.

[0166] Each mock sample is 200 μl. The preparation method of each simulated sample: take 180 μl of healthy human whole blood and 20 μl of Escherichia coli bacteria liquid, mix well, and obtain simulated samples. Prepare multiple mock samples.

[0167] 2. Pre-processing

[0168] Basically the same as step 2 of embodiment 1.

[0169] The only difference is that in step 3, different irradiation times are used for the blue light irradiation of the LED photolysis instrument; 0 means no irradiation.

[0170] 3. DNA extraction

[0171] Take the pre-treated sample obtained in step 2, and use The IndiSpin Pathogen Kit to extract DNA to obtain a DNA sample.

[0172] Four, with the step four of embodiment 1.

[0173] At least three replicate treatmen...

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Abstract

The invention discloses a method for efficiently removing host nucleic acid in a blood flow infection sample. The method can improve the pathogen detection rate. The invention provides a method for removing host nucleic acid in a blood flow infection sample, which is characterized in that the blood flow infection sample is sequentially subjected to the following steps: (1) adding saponin for cracking, and then collecting precipitate; (2) washing by adopting a NaCl solution with the NaCl concentration of more than 0.5 M; (3) adding an azido propyl bromide ingot, carrying out photolysis, and then collecting precipitate; and (4) washing with a NaCl solution with the NaCl concentration of more than 0.5 M. According to the method, host nucleic acid can be effectively removed, so that pathogenic microorganisms in a sample can be more effectively detected. The invention has important application value for identifying blood flow infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for efficiently removing host nucleic acid in bloodstream infection samples, which can improve the detection rate of pathogens. Background technique [0002] Sepsis and bacteremia are collectively referred to as bloodstream infection. Septicemia is a blood infection caused by various pathogenic microorganisms (bacteria or fungi) and toxins invading the bloodstream. The main clinical manifestations are: sudden chills, high fever, tachycardia, shortness of breath, rash, hepatosplenomegaly and A series of serious clinical symptoms such as mental and mental changes, severe cases can cause shock, disseminated intravascular coagulation (DIC) and multiple organ failure. If the bacteria only enter the blood for a short time without clinically obvious symptoms of toxemia (such as blood vessel-related infection), it is called bacteremia. [0003] Bloodstream infection (BSI) has a high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806
Inventor 刘鹏张磊黄文华孔德聪江华律清宇郑玉玲姜永强
Owner ACADEMY OF MILITARY MEDICAL SCI
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