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Method for designing species specific primer for detecting species with known genome information in microbial community and method for measuring bacterium content

A microbial community and primer design technology, applied in the field of determining the content of the corresponding bacterial species in the flora, and the design of species-specific primers, can solve the problems of lack of effective use of genomic information

Inactive Publication Date: 2016-12-14
HARBIN INST OF TECH AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by primer design and detection methods, the current quantification of microbial flora structure can achieve quantification at the classification level of phylum and class, but it lacks the effective use of known genomic information to achieve classification below class by designing specific primers and qPCR quantification A method for the quantification of microbial community structure at the level

Method used

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  • Method for designing species specific primer for detecting species with known genome information in microbial community and method for measuring bacterium content
  • Method for designing species specific primer for detecting species with known genome information in microbial community and method for measuring bacterium content
  • Method for designing species specific primer for detecting species with known genome information in microbial community and method for measuring bacterium content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 6

[0040] Example 1 16S rRNA gene TA clone sequencing and bioinformatics analysis

[0041] First, 200 mg of a soil sample was freshly weighed, and the genome was extracted using the Solarbio Soil Genome Kit or the Shanghai Sangon EZUP Column Soil Genome Extraction Kit, and the final elution volume was 100 μL. Genome template samples (10-50ng / ul) were refrigerated at -80°C for later use. Soil samples can be sealed in plastic bags and stored in a -80°C refrigerator for 1-2 years. Through the amplification of 16S rDNA in a soil sample, the amplification primers are 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACTT-3'). Augmented templates were metagenomics extracted from soil samples. After the PCR amplification product was purified, it was connected to the pMD-19T vector by TA cloning and transformed into competent DH5α Escherichia coli, and then the blue and white spots were screened by LBA culture, and the white spots were picked for sequencing, and ...

Embodiment 2

[0045] Embodiment two specific primer design and screening

[0046] According to the microbial species obtained in Example 1, the name of each microbial species is entered into the EBI genome website (www.ebi.ac.uk / genomes / ) to check whether it has (full) genome information (Table 1), and it ( The whole) genome sequence information was downloaded for subsequent species-specific primer design. The obtained (full) genome sequence information was used as a template for species-specific primer design on the Primer-Blast website (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Enter the (full) genome information of the target strain in the PCR Template box. Since the maximum sequence length here is 50,000bp, only 50kb can be entered each time; the annealing temperature is 60±3℃, and the primer length is 100-400bp. In the "Primer Pair Specificity Checking Parameters" box, select: Automatic, nr, and enter the names or taxonomy ids of all non-target species in the microbial communi...

Embodiment 3

[0053] Configuration and qPCR quantitative determination of embodiment 3qPCR system

[0054] 1. The qPCR system can use a general qPCR kit. Taking GREDBIO's NPK62 kit (including 2×buffer) as an example, see Table 3 for the composition of the qPCR system.

[0055] Table 2 qPCR system composition of NPK62 kit (GREDBIO)

[0056] 2×PCR buffer (containing fluorescent dye and PCR booster)

6μL

Primer 1 (2 μM)

1μL

Primer 2 (2 μM)

1μL

Template+H 2 o

3.8μL

Taq enzyme (5U / μL)

0.2 μL

12 μL total volume

[0057] 2. Preparation of standards: In this experiment, qPCR uses a plasmid containing the target PCR product as a standard.

[0058] The preparation method is as follows: 7 PCR products are gel-cut and purified, connected to pMD-19T vector by TA cloning, and then transformed into competent DH5α Escherichia coli cells. Then the Escherichia coli engineering bacteria were shaken in liquid culture, and the plasmid ...

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Abstract

The invention relates to a method for designing a species specific primer for detecting the species with known genome information in a microbial community and a method for measuring the bacterium content. The designing method is based on a species specific primer of some species with known genome information in a microbial community. The method comprises the following steps: cloning and sequencing a nearly full-length ribosomal gene library; carrying out biological information analysis (BLAST, etc.) to determine the species in a bacterial community and relative abundance of the species; inquiring the species with known genome sequences; based on a PRIMER-BLAST tool, designing a species specific primer, and detecting the specificity of the primer. The bacterium content is measured through a QPCR method: the provided species specific primer is used to quantitatively measure the content of corresponding species in a microbial community. The provided method for measuring the content of known microbes in a microbial community can trace and inspect the number change of some important microbial species in an important biological process (liquor fermentation, for example) in the species level. The provided method can measure the copy number of non-ribosomal gene sequence of species specificity in some microbial genome and cannot obtain the specific number of cells of species in a microbial community.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for designing species-specific primers for determining species with known genome information in the flora and a method for using the primers to measure the content of corresponding strains in the flora. Background technique [0002] At present, the research on species identification in microbial communities focuses on two directions: one is to study the conserved ribosomal gene sequence (such as: 16S, ITS sequence) as the research target, and look at the composition of the microbial community from the overall level; Species microorganisms are the research target, which involves the interpretation of the genome information of species through separation and purification, morphological and molecular biology identification, or de novo metagenomic sequencing. In general, it is obviously inaccurate for the former to determine species information only through conserved sequences, w...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6851C12Q1/6895C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 张会敏李凯强苏立宇何鸿魁余秀娟曹润杰汤知辉王伟民张治洲
Owner HARBIN INST OF TECH AT WEIHAI
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