A kind of primer and quantitative detection method for detecting Neosphingosine resiniferous bacteria

A quantitative detection method and technology of sphingosine bacteria, which are applied in the field of primers and quantitative detection for the detection of neosphingosine bacterium, can solve the problem of heavy workload, limited research and application of sphingosine resiniferous bacteria, and insufficient detection quantity. Accuracy and other issues

Active Publication Date: 2018-04-17
SHANDONG AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] At present, the detection methods for Neosphingosine resiniferous bacteria in the environment mainly rely on isolation and cultivation plus molecular identification. This method has a long counting period, heavy workload, and inaccurate detection quantity, etc., thus limiting the quality of Neosphingosine resiniferous bacteria. Further Research and Application of Bacteria in Remediation of Environmental Problems

Method used

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  • A kind of primer and quantitative detection method for detecting Neosphingosine resiniferous bacteria
  • A kind of primer and quantitative detection method for detecting Neosphingosine resiniferous bacteria
  • A kind of primer and quantitative detection method for detecting Neosphingosine resiniferous bacteria

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Effect test

Embodiment 1

[0058] Example 1, primer design and specificity verification

[0059] 1. Primer sequence design

[0060] According to the differences in the conserved sequences of the 16S region of the rDNA of Neosphingobacterium neosphingobacterium and its close relatives registered in GenBank, and according to the principles of primer design, primers were designed using the primer design software Premier 5.0. Then, the primer fragment sequences were compared with the primer specificity in the NCBI database, and a pair of primers NF and NR were screened out. According to the PCR amplification effect and primer amplification specificity of this pair of primers, this pair of primers was selected for the establishment of a molecular detection technology system for Novosphingobium resinovorum.

[0061] 2. Primer specificity verification

[0062] Respectively, Novosphingobium resinovorum (CGMCC 1.3745), underground neosphingobium (Novosphingobium subterraneum, CGMCC1.3516), extracellular polyme...

Embodiment 2

[0069] Embodiment 2, construction and sample analysis of N. resiniferans plasmid standard

[0070] The bacterial DNA extraction kit (DP302-02) from TIANGEN Company was used to extract the DNA of Neosphingobacterium resiniferans. The extracted DNA was amplified by PCR using the specific primer NF / NR of N.

[0071] The amplification reaction system is 50 μL, containing 5 μL of 10×PCR buffer, 2.5mM Mg 2+ 4 μL, 4 μL of dNTPs (2.5 mM each), 1 μL of upstream and downstream primers (10 mM), 2 U of Taq DNA polymerase.

[0072] The amplification reaction program was: pre-denaturation at 94°C for 5 min, deformation at 94°C for 1 min, annealing at 62°C for 30 s, extension at 72°C for 40 s, a total of 30 cycles, and finally extension at 72°C for 10 min.

[0073] The PCR amplified products were detected by 1% agarose gel electrophoresis, and purified by TIANGEN gel recovery kit, and the purified products were connected to the PMD18T vector of Takara Company overnight at 16°C. The ligate...

Embodiment 3

[0082] The known bacterial concentration is 0.48×10 9 Add 100uL of N. resiniferans in CFU / mL to 0.25g soil and mix evenly, so that the soil contains 1.92×10 N. resiniforans 8 CFU / g, the soil without N. resiniferans was set as the control. Utilize TIANGEN soil kit to extract soil DNA, carry out fluorescent quantitative PCR detection according to the condition of embodiment column 2, the result shows as follows Figure 5 As shown, by detecting the Ct value of the sample against the standard curve, the soil added with N. resiniferans can detect that the copy number of N. resiniforans in the soil sample is 1.59×10 8 copy / g, while N. resiniferous bacteria were not detected in the control soil.

[0083]

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Abstract

The invention relates to a primer and a quantitative determination method for detecting novosphingobium resinovorum. The primer for detecting the novosphingobium resinovorum includes one pair, namely a forward primer and a reverse primer respectively represented in nucleotide sequences SEQ ID NO. 1 and SEQ ID NO. 2. The method also discloses a method for quantitative determination of novosphingobium resinovorum by virtue of the primer. Through real-time fluorescence quantification polymerase chain reaction (PCR) technology, the method disclosed by the invention is applicable to the rapid and quantitative determination of the novosphingobium resinovorum in an environment, so as to provide a rapid and accurate research method for the research of bioremediation of aromatic hydrocarbon pollution.

Description

technical field [0001] The invention relates to a primer and a quantitative detection method for detecting Novosphingobium resinovorum, belonging to the technical field of biological detection. Background technique [0002] Polycyclic aromatic hydrocarbons (PAHs for short) refer to hydrocarbons containing two or more benzene rings in their molecules, which are generally cytotoxic, teratogenic and carcinogenic. Due to the stability of its chemical structure and strong hydrophobicity, it is determined that this kind of compound can exist stably in the environment for a long time, and it is difficult to treat it by conventional methods. With the development of industrial technology, a large number of PAHs enter the environment, and these PAHs are likely to endanger human health through the enrichment of the food chain. The research on the use of microorganisms to degrade polycyclic aromatic hydrocarbons (PAHs) and other persistent organic pollutants has become a development di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2545/114C12Q2531/113
Inventor 周波马艳芳陈升富毛志泉王冰张铭铄束怀瑞
Owner SHANDONG AGRICULTURAL UNIVERSITY
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