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Method for specifically separating sphingomonas yanoikuyae by combining streptomycin resistance surface plate based on special primer polymerase chain reaction (PRC)

A sphingomonas, resistant plate technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of small detection spectrum, inability to amplify, amplify sphingosine Alcoholmonas and other issues

Inactive Publication Date: 2012-08-22
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are a few foreign reports that have attempted to design sphingomonas 16S rDNA specific primers or probes, including degenerate primers or non-degenerate primers, but the designed primers have poor detection specificity, that is, in addition to amplifying sphingosine In addition to Sphingomonas, strains of other genera can be amplified; or the detection spectrum is small, and most of the Sphingomonas strains cannot be amplified
Therefore, there is currently no suitable pair of non-degenerate primers that can specifically amplify the 16SrDNA of Sphingomonas

Method used

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  • Method for specifically separating sphingomonas yanoikuyae by combining streptomycin resistance surface plate based on special primer polymerase chain reaction (PRC)
  • Method for specifically separating sphingomonas yanoikuyae by combining streptomycin resistance surface plate based on special primer polymerase chain reaction (PRC)
  • Method for specifically separating sphingomonas yanoikuyae by combining streptomycin resistance surface plate based on special primer polymerase chain reaction (PRC)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The petroleum-contaminated soil in the Shenfu irrigation area was collected, passed through a 1mm sieve, and stored in a refrigerator at 4°C. Weigh 5g of the soil sample and place it in a sterilized Erlenmeyer flask filled with glass beads, add 45mL of physiological saline, and shake at 150rpm for 2h. Then the suspension was left to stand, the supernatant was taken, and the soil suspension was diluted 10 times with physiological saline. Take 0.1ml to dilute to 10 -2 -10 -6 The soil suspension was spread onto the LB solid medium plate added with streptomycin, and the final concentration of streptomycin was 50 μg ml -1 , 30°C inverted culture for 4 days. Pick the yellow colony grown on the LB streptomycin resistance plate, and repeatedly streak on the LB streptomycin resistance plate until a single colony is isolated.

[0055] LB liquid medium formula is: tryptone 10g, yeast extract powder 5g, NaCl10g, distilled water to 1000ml, pH7.2-7.4; LB solid medium formula is: ...

Embodiment 2

[0058] The PCR product of the correct fragment size amplified in Example 1 was genetically typed by three enzyme digestions, enzyme digestion reaction system (20 μl): 0.5 μl each of the three endonucleases, 2 μl 10× buffer, 0.5 μl bovine serum Albumin (BSA), 2 μl PCR product, 14 μL ultrapure water. The total reaction volume was 20 μl, at 37°C, in a water bath for 2 hours, and 2 μl of 10×loading buffer was added to terminate the digestion reaction. The digested products were separated by 10% polypropylene gel electrophoresis at 90V for 1h. Different restriction enzyme fingerprints can represent different strain genotypes.

[0059] Select a representative strain from each of the above genotypes, and use the bacterial universal primer 27f / 1492r to amplify the full-length sequence of 16SrDNA. The primer sequences are, respectively, 27f: 5'-AGAGTTTGATC[C / A]TGGCTCAG-3'; : 5'-TACGG[A / T / C]TACCTTGTTACGACTT-3'. The PCR reaction system used was the same as in Example 1; the PCR reacti...

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Abstract

The invention relates to the cross field of a molecular biotechnology and a microbiological technique, in particular to a method specifically separating sphingomonas yanoikuyae by combining a streptomycin resistance surface plate based on special primer polymerase chain reaction (PRC). The method comprises the following step: coating a suspension solution of a sample to be detected on a lysogeny broth (LB) culture medium fixed flat plate; selecting yellow bacterial colony; repeatedly scribing to separate purity; extracting pure bacterial strain gene group DNA after being cultured by liquid; amplifying a 16SrDNA segment by adopting the sphingomonas special primer PCR; and carrying out positive amplification and amplification to obtain a segment of 504bp PCR product which is sphingomonas. With adoption of the method, the culturable sphingomonas can be directly and specifically separated from the soil; special species are counted; and a bacterial strain resource of the culturable sphingomonas can be obtained.

Description

technical field [0001] The invention relates to the cross field of microbiology technology and molecular biology technology, specifically a method for specifically isolating Sphingomonas based on genus-specific primer-PCR combined with streptomycin resistance plate. Background technique [0002] Sphingomonas belongs to α-Proteobacteria, Sphingomonales, Sphingomonaceae, because of its certain physiological characteristics (such as containing large plasmids, degrading polycyclic aromatic hydrocarbons , produce yellow pigment, etc.) have previously been assigned to Pseudomonas (Pseudomoans) or Flavobacterium (Flavobacterium). In 1990, Japanese scholar Yabuuchi et al. proposed for the first time that this is a new bacterial genus—Sphingomonas by studying the nucleotide sequence of 16SrRNA, the special glycosphingolipids and the main types of coenzyme Q in intracellular lipids. Fungi (Sphingomonas). The strains of this genus are all Gram-negative bacteria, without spores, move ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 李慧周丽莎张颖徐慧韩斯琴史荣久杨伟超
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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