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Fast nucleic acid extraction, sequencing and identification method based on bacterial 16S rDNA sequence

An identification method and nucleic acid technology, which are applied in the field of rapid nucleic acid extraction and sequencing identification based on bacterial 16S rDNA sequences, can solve the problems of long extraction time, inconvenient steps, limited application scope, etc., and achieve the effect of longer solution time.

Inactive Publication Date: 2020-05-15
北京睿博兴科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1) Disadvantages of kits for extracting bacterial genomes: high cost, low throughput, and long extraction time;
[0008] 2) The methods for PCR amplification of bacterial genomes extracted by some alkaline lysis methods reported in the literature are not simple enough, and the scope of application is also limited;
[0009] 3) The conventional boiling method is difficult to effectively destroy the cell wall of Gram-positive bacteria, which makes the extraction efficiency of genomic DNA extremely low and cannot be used for PCR detection

Method used

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  • Fast nucleic acid extraction, sequencing and identification method based on bacterial 16S rDNA sequence
  • Fast nucleic acid extraction, sequencing and identification method based on bacterial 16S rDNA sequence
  • Fast nucleic acid extraction, sequencing and identification method based on bacterial 16S rDNA sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] DNA extraction from samples

[0062] The sample is the plate streak bacteria or culture of the bacterial strain to be identified;

[0063] Add the plate streaked bacteria or culture into the test tubes, add 20uL cell lysate to each test tube, shake and mix well, let stand at room temperature for 10min-30min, then dilute 10-50 times, shake and mix again, and then high-speed Centrifuge for 2 minutes, and take the centrifuged supernatant as the DNA template to be detected.

[0064] In addition, the cell lysate is a mixture of sodium hydroxide solution and sodium dodecyl sulfate solution, wherein the mass concentration of sodium hydroxide solution is 0.1M-0.5M, wherein the mass percentage of sodium lauryl sulfate solution The concentration is 1%-3%.

Embodiment 2

[0066] PCR amplification reaction

[0067] The general primers are as follows:

[0068] The upstream primer 27F of the universal primer is shown in SEQ ID No.1,

[0069] The downstream primer 1492R of the universal primer is shown in SEQ ID No.2.

[0070] The reaction system of the PCR amplification reaction is calculated as 30uL:

[0071]

[0072] The amplification program of the PCR amplification reaction is:

[0073]

[0074] Repeat for 25 cycles;

[0075] Extend at 72°C for 7 minutes;

[0076] Stop the reaction at 4°C and store.

[0077] It should be noted that the sequences of the universal primers are as follows:

[0078] Forward primer 27F (SEQ ID No.1): 5'-AGAGTTTGATCCTGGCTCAG-3',

[0079] Reverse primer 1492R (SEQ ID No. 2): 5'-TACGGCTACCTTGTTACGACTT-3'.

Embodiment 3

[0081] Analysis of PCR amplification products

[0082] The specific method is as follows:

[0083] (A) Take PCR amplification product, prepare agarose gel and carry out electrophoresis, the voltage of electrophoresis is 100V, after electrophoresis finishes, use gel imager to observe, take pictures and record experimental results;

[0084] (B) cutting out the nucleic acid amplification product of the agarose gel after electrophoresis in step (A), extracting and purifying the nucleic acid;

[0085] (C) The nucleic acid purified in step (B) is used to obtain a spliced ​​sequence using a general-purpose sequence splicing software, and then the spliced ​​sequence is subjected to PCR amplification reaction, and the product of the PCR amplification reaction is sequenced and sequence spliced ​​to obtain 16S rDNA sequence;

[0086] (D) importing the DNA sequence of the corresponding fragment in step (C) into a standard nucleic acid sequence database for sequence comparison to identif...

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Abstract

The invention discloses a fast nucleic acid extraction, sequencing and identification method based on a bacterial 16S rDNA sequence, and belongs to the technical field of nucleic acid sequencing and identification methods. The method comprises the following steps of (1) extracting DNA in a sample; (2)performing PCR amplification reaction in a PCR reaction kit by using the DNA extracted in the step(1) as a DNA template to be detected and using a universal primer for 16S rDNA sequence amplification; and (3) analyzing a PCR amplification product. A bacterial genome crude extraction method with the advantages of wide application range, high flux, high positive detection rate and low cost is provided, and is used for PCR amplification of bacterial 16S rDNA; then, sequencing and strain identification are performed; the whole operation is fast, simple and convenient; the requirements on equipment are low; the bacterial 16S rDNA full-length sequence can be amplified; gram-positive bacteria and gram-negative bacteria can be covered; the identification success rate is high; the strain quantity requirement is low; in addition, the flux is high; and the method can be used for treating hundreds of samples at the same time.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid sequencing and identification methods, in particular, relates to a rapid nucleic acid extraction, sequencing and identification method based on bacterial 16S rDNA sequences. Background technique [0002] 16S rDNA identification refers to the identification of bacterial species by using bacterial 16S rDNA sequence sequencing, which is a method for quickly obtaining bacterial species information. It includes bacterial genome DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing, sequence comparison and other steps. [0003] Bacterial rRNA (ribosomal RNA) is divided into three types according to the sedimentation coefficient, namely 5S, 16S and 23S rRNA. 16S rDNA is the DNA sequence corresponding to the 16S rRNA encoded on the bacterial chromosome. 16S rDNA is accepted by bacteriologists and taxonomists because of its moderate size, abou...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/689C12Q1/04
CPCC12Q1/6869C12Q1/689C12Q1/04C12Q2531/113C12Q2565/125
Inventor 翟晓红韩显徐文彦徐换陈天明
Owner 北京睿博兴科生物技术有限公司
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