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Primer group and method for identifying streptomyces albidoflavus W68

A technology of Streptomyces albus and primer set, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection to achieve high detection efficiency, high sensitivity, and strong specificity

Active Publication Date: 2021-09-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA molecular marker technology is often used in the diagnosis and prognosis of diseases and the screening of pathogenic microorganisms in clinical medicine. It is often used in the identification of plant varieties or resources and the study of species diversity in agriculture. There is no report on the research of tracing the source or tracking its whereabouts through the technology of DNA molecular markers

Method used

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  • Primer group and method for identifying streptomyces albidoflavus W68
  • Primer group and method for identifying streptomyces albidoflavus W68
  • Primer group and method for identifying streptomyces albidoflavus W68

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] material method

[0034] Streptomyces albidoflavus W68, isolated from marine sludge, is a strain of Streptomyces that has a significant biocontrol effect on plant fungal diseases and can be used in microbial fertilizers or microbial pesticides. It was deposited on March 14, 2016 at General Microbiology Center of China Committee for the Collection of Microbial Cultures, the preservation number is CGMCCNo.12210, and the address of the depository unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The depository unit is referred to as CGMCC.

[0035] The genome sequence of Streptomyces albicans W68 was submitted to the NCBI database on November 19, 2020, and the GenBank number is: CP064783.1. The genome size of this strain is 6796629bp.

[0036] The PCR amplification system is: 2uL of 10×Taq Buffer, 1.6uL of 2.5mM deoxynucleotide triphosphates (dNTPs), 0.8uL of 10uM forward primer and 0.8uL of reverse primer, DNA polymerase (TaqDNA polymerase) 0.2uL, te...

Embodiment 2

[0045] Screening of DNA Molecular Markers from the Genome Sequence of Streptomyces albus W68

[0046] Based on the complete genome sequence of Streptomyces albus W68, the following steps were taken to screen for specific sequences:

[0047] Cut the genome sequence into 300bp window bins;

[0048] Compare the obtained bins sequence with the bacterial genome database;

[0049] Filter out bins whose alignment length is greater than 100bp and whose homology is greater than 80%;

[0050] Merge the filtered continuous bins.

[0051] Through the above steps, 232 nucleic acid sequence fragments of different lengths were obtained;

[0052] Further, the above 232 nucleic acid sequences were artificially screened to obtain 10 target sequences for verification;

[0053] The primer6 software was used to design primers for the above 10 nucleic acid sequences, PCR amplification was performed on different templates, and finally the following two specific nucleic acid fragments were screen...

Embodiment 3

[0055] Design and detection of sequence 1751401-1751700 specific primers

[0056] Using the primer6 software, after inputting the nucleic acid sequence fragment 1751401-1751700, the primer sets P1751437F and P1751612R were obtained, and the relevant information of the primers is shown in Table 2

[0057] Table 2: Indexes of primer sets P1751437F and P1751612R

[0058]

[0059] After adding the primer sets P1751437F and P1751612R into the PCR amplification system in Example 1 according to the ratio, the general PCR amplification reaction was carried out according to the PCR amplification procedure in Example 1.

[0060] Such as figure 1 As shown, M stands for Marker, and its fragment size from top to bottom is 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp; the lane number 1 is ddH 2 O is the template, no amplified product was detected; the lane number 2 is based on the genome of Streptomyces alboflavinus W68 as the template, after PCR amplification reaction, the length c...

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Abstract

The invention belongs to the technical field of primer design, and discloses a primer group. The primer group is P2939123F and P2939374R and / or P1751437F and P1751612R. The primer group provided by the invention is high in specificity, high in sensitivity and high in detection efficiency, and can be used for effectively identifying streptomyces albidoflavus W68.

Description

technical field [0001] The invention belongs to the technical field of primer design. Background technique [0002] After the target microorganisms are released into the natural environment, the existence status or dynamic changes of the target microorganisms are monitored or traced from the environmental samples. At present, it is widely used to artificially implant a DNA gene segment in the target microorganism genome as a marker, and then through Detection by means of genetic engineering. The most commonly used gene marker technologies include various fluorescent gene markers, antibiotic gene markers, and auxotrophic gene markers. However, the strains marked by these methods belong to genetically engineered strains, and there are potential safety hazards after being released into the natural environment, which is still controversial at home and abroad. Therefore, searching for specific nucleic acid sequences from the target microorganism's own genome sequence as a molec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/465
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 张振颖李少杰孙宪昀胡成成王旌吉
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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