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Reference product for detecting pathogenic microorganisms of bloodstream infection and preparation method thereof

A technology for detection of pathogenic microorganisms and infections, which is applied in the field of reference products for the detection of pathogenic microorganisms in bloodstream infections and its preparation, can solve problems such as the inability to evaluate sequencing platforms and detection methods, lack of reference materials for pathogenic microorganisms in bloodstream infections, etc., and achieve stable sources Reliable, avoiding experimental errors, and accurate value setting

Active Publication Date: 2021-08-13
湖南赛哲智造科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the clinical application of next-generation sequencing technology in pathogenic infection has only been clinically approved in the past one or two years, there is currently a lack of unified bloodstream pathogenic microbial infection reference materials as reference and operating standards in foreign countries, and it is impossible to carry out different sequencing platforms and detection methods Therefore, there is an urgent need for a bloodstream infection pathogenic microorganism detection reference product that covers various pathogenic bacteria and is close to human natural plasma

Method used

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  • Reference product for detecting pathogenic microorganisms of bloodstream infection and preparation method thereof
  • Reference product for detecting pathogenic microorganisms of bloodstream infection and preparation method thereof
  • Reference product for detecting pathogenic microorganisms of bloodstream infection and preparation method thereof

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preparation example Construction

[0042] The invention discloses a method for preparing a reference product for bloodstream infection pathogenic microorganism detection that covers various pathogenic bacteria and is close to human natural plasma. The specific steps are as follows, and the process is as follows: figure 1 Shown:

[0043] S1: Extract the genomic DNA of cultured human cells and pathogenic microorganisms respectively; if the pathogenic microorganisms cannot be cultured, the target fragment can be synthesized artificially, and then a 200bp pathogenic microorganism-specific fragment can be obtained by PCR amplification.

[0044] S2: Fragmentation of genomic DNA of human cells and pathogenic microorganisms by restriction enzyme digestion.

[0045] The fragmentation enzyme is Fragmentase from NEB Company, and other commercial enzymes with equivalent effects can also be used, such as Yisheng OnePot, etc.;

[0046] S3: Recovery and purification of fragmented DNA.

[0047]The purification magnetic beads...

Embodiment 1

[0054] Example 1 Preparation of fragmented pathogenic microorganism genome DNA reference product

[0055] (1) Genomic DNA fragmentation of human cells and pathogenic microorganisms:

[0056] Three common pathogens of bloodstream infection were selected: Epstein-Barr virus, Pseudomonas aeruginosa, and Candida parapsilosis, representing viruses, bacteria, and fungi, respectively, and Hela cells were used as human cells. The genomic DNA of Hela cells, Epstein-Barr virus, Pseudomonas aeruginosa, and Candida parapsilosis were respectively digested by enzyme digestion, and the enzyme digestion system was prepared according to the following table (20 μl):

[0057]

[0058] Enzyme digestion was carried out at 37°C, and the amount of DNA input and digestion time were optimized as shown in the table below:

[0059]

[0060] After the fragmentation is completed, use XP magnetic beads and 80% ethanol to purify once, and the eluted DNA after purification is used

[0061] Kit dete...

Embodiment 2

[0070] Embodiment 2 Fragmented Genome and PCR Amplified Fragment Mixed Reference Substance Preparation

[0071] Because some pathogenic microorganisms are difficult to obtain pure culture for genomic DNA preparation due to pathogenicity or non-growth in vitro, such as mycoplasma, tuberculosis, rickettsia, mold, etc., so artificial synthesis and PCR are required DNA is obtained by amplification.

[0072] (1) PCR amplified fragments obtained:

[0073] In this example, Mycobacterium tuberculosis, Ureaplasma urealyticum, and Aspergillus terreus were used as representatives. Two species-specific sequences of each pathogen were obtained by bioinformatics means, and then two target fragments were amplified by pathogen-specific primers. The target fragments were both At about 200bp, the pathogen-specific primer sequences are shown in the table below:

[0074]

[0075] Target fragment PCR amplification system and procedures are as follows:

[0076]

[0077] After PCR, the prod...

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Abstract

The invention discloses a reference product for detecting pathogenic microorganisms of bloodstream infection and a preparation method thereof. The preparation method comprises the following steps: (1) respectively obtaining genomic DNAs (Deoxyribose Nucleic Acid) of human cells and pathogenic microorganisms; (2) fragmenting the genomic DNAs of human cells and pathogenic microorganisms by using an enzyme digestion method; (3) recovering and purifying the fragmented DNA; (4) valuing and mixing the fragmented DNA; (5) mixing the fragmented DNA and the plasma; and (6) extraction and quality inspection of the reference product for detecting bloodstream infection. The reference product for detecting the pathogenic microorganisms of bloodstream infection is prepared by using the preparation method. The reference product has a stable source, is close to a natural plasma sample, covers various pathogens, is more accurate in valuing, can be used for performing quality evaluation on a detection result of bloodstream infection detection based on high-throughput sequencing, can be used for comparing results of different sequencing platforms, detection processes and sampling modes, and formulating a technical standard for bloodstream infection detection based on the high-throughput sequencing.

Description

technical field [0001] The invention relates to the technical field of clinical detection of infectious diseases, in particular to a reference product for detection of pathogenic microorganisms of bloodstream infection and a preparation method thereof. Background technique [0002] Bloodstream infection (BSI) refers to the disseminated infection caused by pathogenic microorganisms entering the bloodstream. It is a systemic infectious disease that threatens human life. The main pathogenic microorganisms include bacteria, fungi, and viruses, which can lead to bacteremia and sepsis. Severe cases can cause shock, disseminated intravascular coagulation (DIC), multiple organ failure and even death. Statistics show that BSI accounts for 40% of community-acquired (CA) and hospital-acquired (hospital-acquired HA) sepsis and septic shock cases, and about 20% of ICU-acquired cases. In recent years, with the continuous increase of invasive operations and the unreasonable application of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/686C12Q1/6869
CPCC12Q1/6806C12Q1/686C12Q1/6869C12Q2563/159C12Q2535/122
Inventor 袁光孝林东旭朱方何任胜强陈杰
Owner 湖南赛哲智造科技有限公司
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