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Plasma free DNA marker for bloodstream infection pathogen diagnosis

A DNA library and plasma technology, applied in the field of plasma-free DNA markers, can solve the problems of no corresponding design plan for the size of the amplified fragment product, and high design blindness, and achieve excellent diagnostic timeliness, improved sensitivity, and improved detection performance. Effect

Pending Publication Date: 2020-07-14
深圳华大因源医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the detection process of this method, there is often no corresponding guidance design scheme for the size of the amplified fragment product, and the design is blind. In practical applications, a large number of primers need to be screened and determined.

Method used

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  • Plasma free DNA marker for bloodstream infection pathogen diagnosis
  • Plasma free DNA marker for bloodstream infection pathogen diagnosis
  • Plasma free DNA marker for bloodstream infection pathogen diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1, the research of pathogen free DNA fragment size in plasma

[0082] After bacteria, viruses and other microorganisms infect patients and enter the blood, the pathogens will reproduce and metabolize or be swallowed by white blood cells. After the cells are destroyed, the DNA in the cells will be released into the blood to become plasma free DNA. Therefore, the type of microorganisms can be identified by detecting free microbial DNA fragments in plasma. The nucleic acid sequence of the detected pathogen can be positioned on its reference genome by double-end sequencing, and then the size of its DNA fragment can be calculated. The invention analyzes human-source free DNA and pathogenic free DNA in plasma samples of patients with bloodstream infection, and determines the size of pathogenic free DNA fragments in plasma.

[0083] 1. According to the clinical diagnosis results, select positive plasma samples infected by different pathogen types. The sample inform...

Embodiment 2

[0124] Example 2, the application example of using the plasma cell-free DNA marker established by the present invention for diagnosis

[0125] 1. Select 5 plasma samples whose clinical blood culture was positive for Escherichia coli.

[0126] 2. Take 300 μL plasma sample, and extract plasma free DNA according to the operating instructions of the plasma free DNA extraction kit, and extract two copies of each sample.

[0127] 3. For end repair and adding "A", follow the reaction system shown in Table 8.

[0128] Table 8 Reaction system for end repair and adding "A"

[0129] Reagent Dosage dna 43μL 10×PNK Buffer 5μL 20:1 dATP(25mM) and dNTP(each1.25mM) 1.2μL T4 DNA Polymerase 0.4μL T4 PNK 0.2 μL rTaq 0.2 μL Reaction volume 50μL

[0130] Reaction conditions: 37°C, 30min; 65°C, 15min; 4°C hold.

[0131] 4. Joint connection

[0132] Connect the product obtained in step 3 to the adapter.

[0133] The linker ligatio...

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Abstract

The invention discloses a plasma free DNA marker for bloodstream infection pathogen diagnosis. The invention provides a plasma free DNA treatment method comprising the following step: selecting a fragment with a size of 60-140 bp from to-be-tested plasma free DNA. The method can be used as a sample pretreatment method for bloodstream infection pathogen detection through the plasma free DNA. The distribution range of free nucleic acid in plasma samples infected with Escherichia coli, Klebsiella pneumoniae, staphylococcus aureus, enterococcus faecium and Acinetobacter baumannii is studied, and the distribution of free nucleic acid fragments of the five bacteria in plasma is determined. The plasma free DNA marker has important significance of improving the sensitivity of pathogen detection inplasma metagenome detection and guiding the design of the length range of PCR amplification product fragments to improve the PCR detection sensitivity. The plasma free DNA marker provides a basis forpreparation of pathogen simulation samples in a plasma free DNA detection product development process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a plasma free DNA marker used for pathogenic diagnosis of bloodstream infection. Background technique [0002] Plasma cell-free DNA is a kind of DNA that is free outside the cell. At present, it is believed that the source of cell-free DNA in blood is related to apoptosis, necrosis and cells actively secreting DNA. Under normal physiological conditions, apoptotic and necrotic cells will be removed quickly, so the concentration of free DNA in the blood of healthy people is very low. In the case of malignant tumors, organ transplantation or infection, more free nucleic acids are released and cannot be effectively cleared by the body, so the concentration of free nucleic acids in plasma will increase. [0003] Bloodstream infection is an infection caused by various pathogenic microorganisms invading the bloodstream, including bacteria, viruses, fungi, etc. When a bloodstream infectio...

Claims

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Application Information

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IPC IPC(8): C12Q1/24C12Q1/6869C12Q1/689C12Q1/14C12Q1/10C12Q1/04
CPCC12Q1/24C12Q1/6869C12Q1/689C12Q2535/122C12Q2531/113
Inventor 申奥王晓凤吴红龙
Owner 深圳华大因源医药科技有限公司
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