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Pathogenic microorganism detection method and kit based on metagenomics

A pathogenic microorganism and metagenomics technology, which is applied in the field of pathogenic microorganism detection methods and kits based on metagenomics to achieve the effects of reducing the proportion of small fragments, reducing the loss of DNA and improving the construction efficiency

Active Publication Date: 2020-04-17
天津金匙医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to provide a method for constructing a high-throughput sequencing library based on metagenomics, a method for detecting pathogenic microorganisms, and a kit to solve at least one technical problem in the prior art

Method used

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  • Pathogenic microorganism detection method and kit based on metagenomics
  • Pathogenic microorganism detection method and kit based on metagenomics
  • Pathogenic microorganism detection method and kit based on metagenomics

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0096] In this example, an experiment is carried out on the reaction system of fragmentation enzyme, and the fragmentation enzyme is commercially available DNA Frafmentation Ligase.

[0097] plan a

[0098] DNA sample 1 μl, fragmentation buffer 1 μl, fragmentation enzyme 2 μl, Enhance 0.5 μl, Buffer EB to make up the system to 15 μl,

[0099]

[0100] Option b

[0101] DNA sample 1 μl, fragmentation buffer 2 μl, fragmentation enzyme 4 μl, Enhance 1 μl, Buffer EB to make up the system to 15 μl, and the reaction conditions refer to scheme a.

[0102] plan c

[0103] 1 μl of DNA sample, 1.5 μl of fragmentation buffer, 3 μl of fragmentation enzyme, 0.75 μl of Enhance, and Buffer EB to make up the system to 15 μl, and the reaction conditions refer to scheme a.

[0104] plan d

[0105] 1 μl of DNA sample, 1.5 μl of fragmentation buffer, 5 μl of fragmentation enzyme, 1 μl of Enhance, Buffer EB to make up the system to 15 μl, and the reaction conditions refer to scheme a.

[0...

experiment example 2

[0128] Since polyvinylpyrrolidone K30 (PVP-K30) has a certain cross-linking property at a certain content as an excipient of pharmaceutical preparations, the applicant believes that this cross-linking property may have a positive effect on the homogeneity of the fragmentation reaction.

[0129] Therefore, based on the scheme b in Experimental Example 1, 0.02 mg of PVP-K30 was added to the system to carry out the fragmentation reaction experiment. The results are shown in Table 4:

[0130] Table 4

[0131]

[0132]

[0133] However, compared with the results in Table 2, the results in Table 4 show that the homogeneity after the fragmentation reaction is not improved, but slightly decreased.

[0134] In subsequent experiments, the applicant continued to use scheme b as the basis, adding 0.2 μl polyethylene glycol 2000 and 0.02 mg of PVP-K30 to the system, and carried out the fragmentation reaction experiment. The results are shown in Table 5:

[0135] table 5

[0136] ...

experiment example 3

[0139] In this example, experiments were carried out on the scheme of magnetic bead purification.

[0140] Sample 1: Take the product of Scheme b in Example 1 as Sample 1.

[0141] Sample 2: Based on scheme b in Example 1, 0.2 μl of polyethylene glycol 2000 was added to the system, and the reaction product was sample 2.

[0142] Scenario A, A':

[0143] S1, pipette 10 μL EBBuffer and 30 μL DNA Clean Beads into 20 μL sample, and mix well;

[0144] S2, incubate at room temperature for 1 min;

[0145] S3, after centrifugation, separate the magnetic beads and the liquid under the action of an external magnetic field, and remove the supernatant after the solution is clarified;

[0146] S4, continue to add 100 μL of 70% ethanol solution to rinse the magnetic beads under the action of an external magnetic field, incubate at room temperature for 10 seconds, and remove the supernatant;

[0147] S5, exposing the magnetic beads to air for 5 minutes to dry,

[0148] S6, add 10 μL of ...

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Abstract

The invention provides a pathogenic microorganism detection method and kit based on metagenomics. The method comprises the following steps: extracting nucleic acid; constructing a high-throughput sequencing library; quantifying the sequencing library constructed in the previous step; diluting the quantified sequencing library to 1-4 pM, and carrying out high-throughput sequencing with a sequencer;and analyzing sequencing data obtained in the high-throughput sequencing. The invention further provides a pathogenic microorganism detection kit. The metagenomics-based high-throughput sequencing library construction method and the kit provided by the invention have the advantages that fragmentation enzyme is used for fragmenting DNA, DNA loss is smaller compared with a mechanical method, a fragmentation speed is higher, and the construction efficiency of the whole library is improved.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a method and kit for detecting pathogenic microorganisms based on metagenomics. Background technique: [0002] Infectious diseases are a general term for diseases caused by pathogenic microorganisms (bacteria, viruses, fungi, parasites, etc.). According to statistics, the cause of death from infectious diseases accounts for more than 25% of all causes of death, and is a major disease that seriously threatens human health in the world today. There are about 40 million patients who suffer from infectious diseases every year in the world, and my country's patients account for about 1 / 4, of which the unknown etiology of clinical diagnosis accounts for nearly 50% (about 5 million). When infectious diseases of unknown cause occur, rapid and accurate identification of pathogens is the key to precise treatment, effective surveillance, control of disease spread, and reduction of medical bur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/04C40B50/06
CPCC12Q1/6869C40B50/06C12Q2535/122
Inventor 梁永王棪李立锋任若通蒋智
Owner 天津金匙医学科技有限公司
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