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Library-building sequencing method for detecting full length of bacterial 16S rDNA

A full-length, sequencing technology, applied in biochemical equipment and methods, sequence analysis, chemical library, etc., can solve the problems of inability to accurately determine the ecological structure of environmental microorganisms, inability to detect strain levels, etc., to achieve good fidelity, The effect of accurate ecological structure

Active Publication Date: 2019-11-15
北京群峰纳源健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, it is not possible to fully detect the level of bacterial species, it is not possible to accurately determine the ecological structure of environmental microorganisms, and it is used for the detection of specific bacterial species in samples, such as the detection of pathogenic bacteria and probiotics

Method used

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  • Library-building sequencing method for detecting full length of bacterial 16S rDNA
  • Library-building sequencing method for detecting full length of bacterial 16S rDNA
  • Library-building sequencing method for detecting full length of bacterial 16S rDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Extraction of sample DNA

[0048] 1.1 The present invention uses Qiagen's DNeasy PowerSoil Kit to extract total DNA from a stool sample, which is marked as sample A.

[0049] Specific steps:

[0050] a. Shake the collection tube containing 3ml of protective agent stool samples, divide them into two 1.5ml centrifuge tubes, remove the supernatant at 2500g for 10min, transfer the precipitate to the Power Beads Tube with a 1ml wide-mouth pipette tip, and gently vortex to mix.

[0051] b. Add 60μl C1 and vortex for 10min.

[0052] c. Centrifuge at 10000g for 30s.

[0053] d. Transfer the supernatant to a 2ml collection tube.

[0054] e. Add 250μl C2 to the collection tube, shake for 5s, and incubate at 4°C for 5min.

[0055] f. Centrifuge at 10000g for 1 min.

[0056] g. Transfer 600μl of supernatant to a 2ml collection tube.

[0057] h. Add 200μl C3, shake and mix, and incubate at 4°C for 5min.

[0058] i. Centrifuge at 10000g for 1min.

[0059] j. Transfer 750μl of supernatant to a new...

Embodiment 2

[0133] The DNA samples of four known strains (Streptococcus_pneumoniae, Enterococcus_faecalis, Streptococcus_pyogenes, Streptococcus_agalactiae) were mixed according to the abundance of the flora of 1:1:1:1 to form a mock community, using the 16S-FAST technology of the present invention Detect the fidelity of the sample flora abundance. The result is Image 6 Shown from Image 6 The results show that the fidelity of the bacterial population abundance determined by the technology of the present invention is very good, and is basically close to the true ratio.

Embodiment 3

[0135] Using the method of the present invention to detect the 16S full length of the sample as the experimental group, using the V3V4 segment sequencing detection method in the prior art as the control group, comparing the detection results of the two, as shown in Table 2:

[0136] Table 2 Abundance of the top 20 genus of two methods

[0137] Generic name A-V3V4 A-16S Prevotella60.8760.56 Megamonas19.6121.54 Bacteroides5.855.81 Roseburia3.872.72 Faecalibacterium1.501.89 Enterobacter1.360 Sutterella1.172.07 Ruminococcus1.130.68 Clostridium0.860.01 Dorea0.721.08 Parabacteroides0.690.77 Blautia0.600.37 Coprococcus0.580.02 Eubacterium0.311.43 Gemmiger0.150 Bifidobacterium0.080 Klebsiella0.080.32 Butyricicoccus0.070.03 Leuconostoc0.070 others 0.420.7

[0138] From Table 2 and Figure 7 It can be seen that the correlation coefficient R of the abundance of the genus determined by the two methods 2 Is: 0.998, indicating that the genus categories determined by the two...

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Abstract

The invention discloses a library-building sequencing method for detecting full length of bacterial 16S rDNA. By adding a molecular tag UMI to each sample, a ligation library and a splicing library are separately amplified, the two libraries are sequenced, the data is extracted by recognizing the UMI combination, and the full length sequence of 16S rDNA was assembled, and the species type is determined by comparing the databases. The library-building method of the invention is suitable for the detection of the flora structure of all types of samples, and can upgrade the traditional flora structure identification from a genus level to a species level, compared with the genus level, the species level has the advantage that an ecological structure of environmental microorganisms can be more accurately determined, which facilitates deep research, and method realizes the detection of specific bacterial species in samples.

Description

Technical field [0001] The invention belongs to the technical field of bacterial molecular biology, and specifically relates to a library-building sequencing method for detecting the full length of bacterial 16S rDNA. Background technique [0002] The 16S rRNA gene is a gene unique to prokaryotes and has a very high copy number in prokaryotes. The full-length 1542nt DNA sequence contains 9 spaced hypervariable regions, and the 16S rRNA gene sequence, which is both specific and conservative, is widely used as a microbial marker in research. When researching 16S human DNA before, the method of Sanger sequencing combined with cloning or chip hybridization was usually used, but the traditional method has the disadvantage of low throughput. Many researchers have used 16S rDNA to classify microbes in the environment, fecal microbes, and skin microbes. At present, the second-generation sequencing technology has become the mainstream method of microbial research, but due to the limitat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/04C40B50/06G16B30/10
CPCC12Q1/6869C40B50/06G16B30/10C12Q2531/113Y02A50/30
Inventor 冯涛马伟志王震
Owner 北京群峰纳源健康科技有限公司
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