Construction method and application of microbial diversity library

A library construction and microbial technology, applied in biochemical equipment and methods, microbial measurement/testing, chemical libraries, etc., can solve the problems of sample abundance errors, inability to construct diverse libraries, inability to identify multiple species in the same sample, etc.

Inactive Publication Date: 2018-04-13
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The total amount of DNA extracted from some precious samples and trace samples is small, and even fails to meet the requirements of the kit for DNA extraction. This part of the sample cannot be used for the construction of a diversity library, which limits the application of diversity analysis.
[0011] (2) Multi-species identification cannot be performed on the same sample: Conventional diversity library construction is to perform PCR amplification on the entire group of DNA, and can only amplify and build a library for a certain region such as 16s rDNA, 18s rDNA, and ITS regions
It is impossible to conduct a comprehensive analysis of all samples of multiple species in the sample, such as bacteria, fungi, archaea, etc.
[0012] (3) Strong sample bias: PCR method is used to amplify the conserved sequence region in the construction of conventional diversity library, and the information with low abundance in the sample group is often less or even missing due to the bias of PCR. Accurately reflect the real situation of the sample
[0013] (4) There is an error in the abundance of the sample: the PCR method is used for the construction of the conventional diversity library, and the PCR primers involved are likely to cause deviations due to differences in Tm values, and the relevant reference correction sequences are not added to the primer design, resulting in deviations in the analysis results

Method used

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  • Construction method and application of microbial diversity library
  • Construction method and application of microbial diversity library
  • Construction method and application of microbial diversity library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] 1. Synthesis of special index primer adapter:

[0187] The special index primer adapter is a double-stranded adapter with a sticky end, one end of the special index primer adapter is a first sticky end, and the first sticky end includes at least a special end sequence, and the special end The sequence is used to identify the target region of the microbial genome DNA, and is connected to the single-stranded DNA of the target region through complementary pairing. The target region may be 16S rRNA region, 18S rRNA region or ITS region, etc. The structure of the special end sequence is "optional sequence-recognition target sequence", the optional sequence is used to adjust the Tm of the linker and ensure that the Tm between the linkers is similar, and the recognition target sequence is used for matching with the target region Complementary pairing of nucleotide sequences.

[0188] 1. As an example, use Primer1 and Primer2 to synthesize special index primer adapters,

[018...

Embodiment 2

[0340] (1) Take 10 ng of feces samples and divide them into 6 parts on average, and group them according to Table 14 below. Negative controls were set for each group, named N1&N2 respectively:

[0341] Table 14

[0342]

[0343] Among them, A-C samples were extracted and quantified using picogreen double-strand quantitative dye, and the quantitative results were all lower than the detection limit.

[0344] The general database construction scheme is as follows:

[0345] 1. Prepare the following PCR reaction mixture:

[0346] Table 15

[0347] sample

Volume (ul)

KAPA HiFi HotStart ReadyMix (2x)

25

Primer library (10μM)

2

NF water

23

total capacity

50

[0348] Wherein, the primer library refers to primer pairs well-known to those skilled in the art for amplifying microorganisms such as bacteria, fungi, archaea, denitrifying bacteria, nitrogen-fixing bacteria, nitrite reducing bacteria, ammonia oxidizing bacteria, a...

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Abstract

The invention relates to the technical field of molecular biology and particularly relates to a construction method and an application of a microbial diversity library. According to the novel construction method of the microbial diversity library, the microbial diversity library of a metagenome is constructed by virtue of single chain linkage and a special label primer joint; the scheme is applicable to conventional samples, trace samples, FFPE samples and less than 150 cell groups, and the application range of microbial diversity research is extended; and by introducing the single chain linkage and a special label primer, different target populations fragments can be enriched in a small deviation manner, so that the richness of species and the authenticity of abundance of different species are guaranteed. Besides, the construction method further has the advantages that the initial mass is low, and the same sample can be subjected to multi-species authentication.

Description

technical field [0001] The invention relates to the field of microbial diversity research, in particular to a method for constructing a microbial diversity library and its application. Background technique [0002] Microbial community structure and diversity, as well as microbial function and metabolic mechanism are research hotspots in microbial ecology. For a long time, due to technical limitations, the understanding of the structure and diversity of microbial communities has not been comprehensive, and little is known about microbial functions and metabolic mechanisms. In recent years, sequencing technology has developed rapidly and has become an important means of biological research. The rise of high-throughput sequencing technology has made it possible to study metagenomic sequences and microbial diversity on a large scale and at low cost. The purpose of microbial diversity analysis is to study the types and quantities of microorganisms in the environment, and to rev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12N15/1093C12Q1/6869C40B50/06C12Q2525/191C12Q2533/101C12Q2521/501
Inventor 李静陈昌岳王芳张璐璐胡秋萍张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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