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Method for analyzing and identifying clinical difficult strains by 16S rRNA gene sequence

An analysis method and gene sequence technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, resistance to vector-borne diseases, etc., can solve the problem of low sensitivity, unstable phenotypic expression, automatic bacterial identification instrument Can not give identification results and other problems, to achieve the effect of expanding the identification spectrum and meeting the identification needs

Pending Publication Date: 2020-12-11
AFFILIATED HOSPITAL OF NANTONG UNIV
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Problems solved by technology

However, there may also be the following problems: the dependent phenotypic expression is unstable; the sensitivity is not high, some methods are only applicable to a single strain; the application of some methods is also affected by serovars [3-6]
Therefore, for some difficult and rare strains, the automatic bacterial identification instrument cannot give ideal identification results.

Method used

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  • Method for analyzing and identifying clinical difficult strains by 16S rRNA gene sequence
  • Method for analyzing and identifying clinical difficult strains by 16S rRNA gene sequence
  • Method for analyzing and identifying clinical difficult strains by 16S rRNA gene sequence

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Embodiment 1

[0037] This embodiment discloses a method for identifying difficult clinical strains. Specifically, the 16S rRNA gene sequence analysis method is used, including:

[0038] S1: Extract the bacterial DNA of the strain to be identified, select bacterial universal primers, and establish a PCR method for amplifying bacterial 16S rRNA;

[0039] S2: Optimizing the PCR reaction system and reaction conditions to obtain amplification products;

[0040] S3: Sequence comparison of the amplified product with the gene database to obtain the bacterial species to be identified.

[0041] Specifically, the following steps are included:

[0042] 1. Genomic DNA extraction

[0043] The specimens were from bacteria that could not be identified by conventional methods in the Affiliated Hospital of Nantong University from 2015 to 2019. Take the activated clinical isolate strain 4 and inoculate it on the blood plate, at 35°C, supplemented with 5% CO 2 Colonies were collected after 24-48 hours of a...

Embodiment 2

[0058] This embodiment studies the detection and analysis of some clinical difficult bacterial species, and the results are as follows:

[0059] 2.1 Detection and analysis results of Bordetella holmesii (B.holmesii)

[0060] The total DNA of the isolated bacteria was extracted as a template, amplified using the optimized PCR reaction system and conditions, and the product was bidirectionally sequenced (see figure 2 ), and then carried out BLAST comparison with the Genebank database, the results showed that the 16S rRNA sequence of the isolate was similar to the 16S rRNA sequence of B.holmesii reported in the database, and the similarity reached 99% ( image 3 ), so the strain was identified as B.holmesii. The 16S rRNA gene sequence of the isolate was uploaded to NCBI GenBank with access number KT828544.1. The isolation of B.holmesii has not been reported in China, so the strain was uploaded to the China General Microorganism Culture Collection Management Center (CGMCC, stra...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for analyzing and identifying clinical difficult strains by a 16S rRNA gene sequence. The method for identifying the clinical difficult strains adopts a 16S rRNA gene sequence analysis method, and comprises the following steps: S1, extracting bacterial DNA of a strain to be identified, selecting a bacterial universal primer, and establishing a PCR method for amplifying bacterial 16S rRNA; S2, optimizing a PCR reaction system and reaction conditions to obtain an amplification product; and S3, performing sequence comparison on the amplification product and a gene database to obtain the strain of the strain to be identified. The invention establishes a new method for effectively identifying the difficult strains by applying a 16S rRNA gene sequence analysis technology, expands the identification spectrum of clinical strains, and meets the clinical identification requirements on the difficult strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for analyzing and identifying difficult clinical strains of 16S rRNA gene sequence. Background technique [0002] 16S rRNA gene sequence analysis refers to obtaining the 16S rRNA sequence information of the microorganisms in the sample by cloning, sequencing or enzyme digestion, and probe hybridization, and then comparing with the 16S rRNA database, so as to carry out species identification and evolutionary tree of the microorganisms in the sample Analysis is a fast and effective method. 16S rRNA exists in all organisms, and its evolution has good clock properties, and its structure and function are highly conserved. It is called "bacterial fossil", so 16S rRNA is the most commonly used molecule in the identification of bacterial species and systematic taxonomy. bell [1-2] . [0003] In biological cells, ribosomal RNA (rRNA) combines with dozens of proteins to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/04
CPCC12Q1/686C12Q2537/165Y02A50/30
Inventor 浦江褚少朋汤自洁钱晨李娴
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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