Specific primer capable of being used for real-time-polymerase chain reaction (PCR) amplification streptomyces deoxyribonucleic acid (DNA)

A specific, Streptomyces technology, used in recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc.

Inactive Publication Date: 2012-04-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims to solve the current problem of lacking primers to amplify Streptomyces target sequences from sample DNA by Real Time PCR, and provides a specific primer for amplifying Streptomyces

Method used

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  • Specific primer capable of being used for real-time-polymerase chain reaction (PCR) amplification streptomyces deoxyribonucleic acid (DNA)
  • Specific primer capable of being used for real-time-polymerase chain reaction (PCR) amplification streptomyces deoxyribonucleic acid (DNA)
  • Specific primer capable of being used for real-time-polymerase chain reaction (PCR) amplification streptomyces deoxyribonucleic acid (DNA)

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Experimental program
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Embodiment 1

[0014] Streptomyces (ATCC 13273), Bacillus subtilis (Bacillus subtilis, ATCC 6633), Sphingomonadales (Sphingomonadales, ATCC 27551), Rhodococcus (Rhodococcus, ATCC6939), Burkholderia (Burkholderia, ATCC) 17616) 5 genera bacterial DNAs were used as templates to carry out the specificity verification test of primer VF / VR. Bacteria were purchased from ATCC (American Type Culture Collection).

[0015] The PCR amplification system is: 25μL consisting of 0.5μL DNA template (about 10ng), 0.5μL forward primer SF (10μM), 0.5μL reverse primer SR (10μM), 0.5μL dNTPs (10mmol / L), 2.5μL 10× PCR buffer (with MgCl2), 0.2μl TaqDNA polymerase (5U / μL), sterile double distilled water to make up 25μL. PCR amplification reaction conditions were: pre-denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 45 s, a total of 35 cycles, extension at 72 °C for 5 min, and storage at 4 °C.

[0016] The sequence amplified by this embodiment is d...

Embodiment 2

[0018] Soil DNA was extracted from soil samples from Langfang City, Hebei Province and Wuhan City, Hubei Province with the Soil DNA Kit of OMEGA Company. The primers SF / SR were used to carry out PCR amplification test on 8 extracted soil DNAs. The PCR amplification system is: 25 μL consists of 1 μL DNA template (about 10 ng), 1 μL forward primer SF (10 μM), 1 μL reverse primer SR (10 μM), 12.5 μL, sterilized double distilled water to make up 25 μL. PCR amplification reaction conditions were: pre-denaturation at 95 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 45 s, a total of 35 cycles, extension at 72 °C for 5 min, and storage at 4 °C.

[0019] The sequence amplified by this embodiment is detected by agarose gel electrophoresis, and the detection results are as follows: figure 2 shown, from figure 2 It can be seen that the specific primer SF / SR used to amplify Streptomyces in this experiment can accurately amplify the tar...

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Abstract

The invention belongs to the field of biological detection and relates to a specific primer capable of being used for real-time-polymerase chain reaction (PCR) amplification streptomyces deoxyribonucleic acid (DNA). A forward primer of the specific primer for the amplification streptomyces DNA is shown as SEQ IDNo.1, and a reverse primer SR sequence is shown as SEQ ID No.2. The primer is designed according to the basic group differences of the streptomyces ribosomes rDNA and the real-time-PCR primer requirements. When the primer SF / SR of the invention is applied, streptomyces target fragments with the length being 200bp can be directly and fast amplified from various kinds of sample DNA containing the streptomyces. The primer can be applied to the real-time-PCR, and the streptomyces in the soil can be fast and quantificationally detected.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a specific primer that can be used for Real Time-PCR amplification of Streptomyces DNA. Background technique [0002] Streptomyces is an actinomycetes with a wide range of geographical distribution and host range. It is mainly found in soil and decaying plants. Most Streptomyces produce spores, which are important saprophytes and cause soil to produce spores. Earthy smell. Streptomyces belong to Actinobacteria, about 550 species of Streptomyces have been found, and new species are constantly being discovered. [0003] An important contribution of the Streptomyces family to humans is the production of various antibiotics. Streptomycin, tetracycline, and erythromycin are all produced by them. Streptomyces can tolerate extreme environments in soil. Scientists found from the measured genome sequence that Streptomyces has a large number of genes to adapt to different environments...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11
Inventor 李顺鹏郑婕陈俊辉
Owner NANJING AGRICULTURAL UNIVERSITY
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