A method for identification of microbial populations by two nucleotide real-time sequencing-by-synthesis profiles

A two-nucleotide, synthetic sequencing technology, applied in the biological field, can solve the problems of unfavorable trace template detection and identification, large demand for PCR product samples, increased sequencing steps and sequencing time, etc., to increase the length of sequencing and reduce the cost of sequencing , the effect of reducing the number of reactions

Inactive Publication Date: 2015-07-29
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The former requires a large amount of PCR product samples, which is not conducive to the detection and identification of trace templates
In the latter, product denaturation will correspondingly increase the sequencing steps and sequencing time, and reduce the sequencing efficiency

Method used

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  • A method for identification of microbial populations by two nucleotide real-time sequencing-by-synthesis profiles
  • A method for identification of microbial populations by two nucleotide real-time sequencing-by-synthesis profiles
  • A method for identification of microbial populations by two nucleotide real-time sequencing-by-synthesis profiles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Method for Identifying Microbial Populations Using Two Nucleotide Real-Time Synthesis Sequencing Profiles

[0026] 1.13 Mycobacterium sp. rnpB gene peak spectrum prediction

[0027] With 13 kinds of mycobacteria (M.paratuberculosis, M.tuberculosis, M.gastri, M.kansasii, M.marinur, M.gordonae, M.malmoense, M.leprae, M.intracellular, M.xenopi, M.celatum, M. fortuitum, M. smegmatis) as an example, to describe the method of two nucleotide real-time sequencing peaks to identify microbial populations. In this example, the rnpB gene was selected as the sequencing target. The sequences of the variable regions of the partial rnpB genes of these 13 mycobacteria are shown in Table 1. First, according to the gene sequences of the strains, the peak spectrum information of the partial rnpB gene variable regions of these 13 mycobacteria genera was predicted in the case of two-nucleotide real-time synthetic sequencing.

[0028] Table 1 Partial variable region sequences of...

Embodiment 2

[0037] Example 2: Identification of Two Mycobacteria Based on Pyrosequencing Platform's Two-nucleotide Real-Time Synthesis Sequencing Map

[0038] In order to verify the feasibility of this method in the identification of actual samples, two strains of Mycobacterium paratuberculosis (M.paratuberculosis) and Mycobacterium celaturn (M.celaturn) were identified using two-nucleotide real-time sequencing-by-synthesis. In this example, a pyrosequencing platform based on real-time sequencing-by-synthesis is used.

[0039] (1) DNA sample preparation

[0040]First, the genomic DNA of M. paratuberculosis and M. celaturn strains was extracted and stored at -20°C until use. Part of the rnpB gene (RNasePRNAgene) variable region of the strain was amplified by PCR under the action of polymerase. Amplification primers are: F: 5'-CGGATGAGTTGGCTGGGCGG-3'; R: 5'-GGGTGAAACGGTGCGGTAAGAGC-3'. Among them, the 5' end of the reverse primer was labeled with biotin to obtain the PCR samples required ...

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Abstract

The invention provides a trace microorganism identification method. Two nucleotides are added into a sequencing reaction at the same time to obtain a series of peak-map information graphs. In the peak-map information graphs, different templates need different sequencing reaction times, and the sequencing reaction peak-map signal intensities obtained by different sequencing modes are different. The identification of microbial flora can be realized through the principle. The method effectively improves the sensitivity of microorganism identification, shortens the operation time and provides a reliable choice for microorganism separation and identification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and is a method for identifying microbial populations, in particular to a method for identifying microbial populations by real-time synthesis and sequencing of two nucleotides and its application. Background technique [0002] The identification of microorganisms has a wide range of applications in disease prevention and treatment. Rapid and accurate detection of microorganisms plays an important role in disease infection and antibacterial treatment, especially the detection of a small number of pathogenic microorganisms. For example, drug-resistant strains in the treatment of pulmonary tuberculosis have brought great difficulties to the treatment in recent years, and it is necessary to effectively identify the strains in order to take targeted treatment with individualized drugs. A commonly used method is to perform PCR amplification analysis on the variable regions of phylogenetic markers. At pre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/32C12R1/33
CPCC12Q1/6869C12Q2535/113C12Q2565/301
Inventor 肖鹏峰浦丹
Owner SOUTHEAST UNIV
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