Unlock instant, AI-driven research and patent intelligence for your innovation.

Metagenomics-based library building method and detection kit

A metagenomics and genomics technology, applied in the field of metagenomics-based library construction methods and detection kits, can solve complex, labor-intensive and PCR instrument processes, etc., to reduce error rates, facilitate detection accuracy, reduce The effect of the user's request

Active Publication Date: 2020-11-13
广州微远医疗器械有限公司 +3
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a complex, manpower-intensive and PCR machine-intensive process for medical testing laboratories that need to construct a large number of samples and multiple sample types for metagenomics every day, requiring medical laboratories to invest more manpower and material resources to ensure daily Accurate and timely deliveries work smoothly

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Metagenomics-based library building method and detection kit
  • Metagenomics-based library building method and detection kit
  • Metagenomics-based library building method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] A pathogen detection method, comprising the following steps:

[0056] 1. Sample source.

[0057] The experimental samples are clinically positive samples M1-M18, and the types of pathogens are determined by PCR, culture and other methods as shown in the table below.

[0058] Table 1. Types of pathogens in various types of samples

[0059]

[0060]

[0061] Second, the method.

[0062] 1. Build a database.

[0063] S1: Use high-purity nucleic acid extraction methods to extract all genomic nucleic acids in the clinical samples to be tested. The specific methods are as follows:

[0064] S1.1. Sample cracking:

[0065] Add the sample to be tested into the lysate, mix well, incubate at 70°C for 15 minutes after centrifugation, and cool to room temperature after centrifugation again.

[0066] S1.2, DNA purification:

[0067] Transfer all the lysed lysate to the adsorption column, centrifuge to discard the waste liquid, add buffer, centrifuge to remove the protein ...

Embodiment 2

[0105] A pathogen detection method screening is carried out as follows.

[0106] 1. Method.

[0107] With reference to the method of embodiment 1, the difference is only in:

[0108] 1. In step S2, the input amount of genomic nucleic acid DNA ranges from 100pg to 1μg, and the amount of endonuclease used is still 20mU;

[0109] 2. In step S3, the amount of linker is added according to the amount of DNA input, as shown in the following table:

[0110] Table 6. Input amount of genomic nucleic acid and adapter amount

[0111] DNA input Connector usage 500ng–1μg 40±1ng 100ng–500ng 20±1ng 25ng–100ng 10±1ng 5ng–25ng 2±0.5ng 100pg–5ng 0.2±0.1ng

[0112] Note: The above "adapter usage" is adjusted according to the input amount of DNA nucleic acid.

[0113] 3. In step S5, the number of PCR amplification cycles is adjusted according to the amount of DNA input in the following table:

[0114] Table 7. Genomic nucleic acid input amoun...

Embodiment 3

[0124] A pathogen detection method, carried out with reference to the method in Example 1, the only difference is that according to the amount of DNA input: linker amount=3ng:0.07ng (group 1) or 3ng:0.52ng (group 2) or 3ng:1.36ng ( Group 3), adjust the amount of joints. The results obtained are as follows.

[0125] Table 9. Library Adapter Proportion (ng) of Different Adapter Input Amounts

[0126]

[0127] The results are as above table and Figure 3-4 as shown, image 3 It is a comparison of the library concentration (ng / μL) under different adapter input conditions. When the adapter input amount is 0.07ng, the library concentration is significantly lower than that in Example 1 when the input amount is 0.2ng. Among them, the library concentration of 3 samples is It is much lower than 1ng / μL, and the library concentration of 5 samples is around 1ng / μL. It cannot be satisfied with one pooling of multiple samples at a time or needs to concentrate the samples before pooling...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a metagenomics-based library building method and a detection kit, and belongs to the technical field of infection pathogen diagnosis. The metagenomics-based library building method comprises the following steps: S1, extracting genome nucleic acid in a to-be-detected sample; S2, in an enzyme digestion system, carrying out enzyme digestion fragmentation on endonuclease, andcarrying out terminal repair on the DNA fragment by using terminal repair enzyme; S3, adding a linker and ligase into the solution obtained in the step S2, and performing linker connection in a buffersolution system; S4, purifying the connected product by using magnetic beads; S5, amplifying the purified product; and S6, purifying the amplified product by using magnetic beads, and removing smallfragments. The library building method is suitable for laboratories needing metagenome library building of a large number of samples and nucleic acids of various sample types, the utilization rate ofpersonnel and instruments is increased, the error rate is reduced, the success rate of library building can be guaranteed, and the 24-hour extreme delivery target is achieved.

Description

technical field [0001] The invention relates to the technical field of infection pathogen diagnosis, in particular to a metagenomics-based library construction method and a detection kit. Background technique [0002] Metagenome (mNGS) technology has gradually become a widely used technology in many aspects of discovery and translational research. It has important applications in the pathogenic diagnosis and analysis of urgent and critical infectious diseases. It can conduct unbiased analysis of all nucleic acids in samples. Sequencing, including nucleic acids of human and microbial origin. [0003] In vitro infection diagnosis technology can be used for the detection of clinical samples of all infection types, such as sputum, alveolar lavage fluid, and throat swabs for respiratory tract infections; cerebrospinal fluid for central nervous system infections; blood for bloodstream infections and other sample types, such as pleural and ascites , tissues, paraffin sections, etc...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2521/101C12Q2521/301C12Q2521/501C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 许腾谢淑媚曾伟奇陈海如刘足周晓思李永军王小锐苏杭
Owner 广州微远医疗器械有限公司
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com