Molecular detection method for desulfovibro

A quantitative detection method and sulfate technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of the lack of development of sulfate-reducing bacteria detection methods and high detection costs, and achieve The effect of short detection time, high sensitivity and low detection cost

Active Publication Date: 2015-12-02
北京世纪金道石油技术开发有限公司 +1
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  • Application Information

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Problems solved by technology

This method is fast, sensitive and highly specific for specific sulfate-reducing bacteria. However, the detection cost is high, and currently it

Method used

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  • Molecular detection method for desulfovibro
  • Molecular detection method for desulfovibro
  • Molecular detection method for desulfovibro

Examples

Experimental program
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Effect test

Embodiment 1

[0043] The establishment of standard curve and linear equation of the quantitative detection of embodiment 1 sulfate-reducing bacteria

[0044] 1. Test method

[0045] 1.1 Sample collection and processing

[0046] The sulfate-reducing bacteria reagent bottle was used to selectively cultivate sulfate-reducing bacteria in the oil well production fluid of a certain block of Liaohe Oilfield at 55°C. After seven days, the concentration of sulfate-reducing bacteria in the reagent bottle was detected to be 1×10 8 per ml, make the dilution gradient shown in Table 1.

[0047] Table 1 Bacterial solution dilution concentration and corresponding relative brightness value

[0048]

[0049] 1.2 Pretreatment of samples

[0050] 1. Take 1ml of the diluted bacterial solution in a 1.5ml centrifuge tube, centrifuge at 12000rpm for 10min, and discard the supernatant.

[0051] 2. Add 100 μL lysozyme to treat at 37°C for half an hour, use 20mM Tris for lysozyme, pH=8; 2mM Na 2 -EDTA buffer ...

experiment example 1D

[0062] Experimental Example 1 Determination of the Lower Limit of the DNA Template Concentration

[0063] 1. Experimental method

[0064] 1.1 Sample collection and processing

[0065] Sulphate-reducing bacteria in the oil well production fluid of a block in Liaohe Oilfield were taken, and cultured in a SRB reagent bottle at 55°C for 7 days.

[0066] 1.2 DNA extraction (for the extraction method, refer to the instruction manual of the Genome Extraction Kit of Tienensis Bacteria)

[0067] The concentration of DNA detected by NanoDrop2000 was 160ng / μL.

[0068] 1.3PCR amplification

[0069] With the extracted bacterial DNA as a template, configure the PCR amplification system according to Table 2 to amplify (ddH 2 Add after O dilution), wherein, the primer pair used is that the forward primer shown in SEQIDNo.1 and the nucleotide sequence are made up of the reverse primer shown in SEQIDNo.2 by nucleotide sequence; Wherein, Y represents base C Or T, R represents the base A or...

experiment example 2

[0078] Experimental example 2 Determination of the lower limit of the concentration of sulfate-reducing bacteria

[0079] 1. Test method

[0080] 1.1 Sample collection and processing

[0081] The sulfate-reducing bacteria reagent bottle was used to selectively cultivate sulfate-reducing bacteria in the oil well production fluid of a block in Karamay Oilfield, Xinjiang at 37°C. After seven days, the sulfate-reducing bacteria concentration in the reagent bottle was detected to be 2×10 5 pieces / ml. For the cultured sulfate bacteria, do the dilution shown in Table 3.

[0082] Table 3 Dilution concentration of bacterial solution

[0083]

[0084] 1.2 Pretreatment of samples

[0085] 1. Take 1ml of the diluted bacterial solution in a 1.5ml centrifuge tube, centrifuge at 12000rpm for 10min, and discard the supernatant.

[0086] 2. Add 100 μL lysozyme to treat at 37°C for half an hour, use 20mM Tris for lysozyme, pH=8; 2mM Na 2 - The EDTA buffer solution is prepared with a fina...

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Abstract

The invention discloses a molecular detection method for desulfovibro. The nucleotide sequences of the specific primer pair adopted by the detect method are SEQ ID No.1 and SEQ ID No.2. The detection method comprises the following steps: (1) establishing a standard curve and a linear equation for quantitative detection of desulfovibro; (2) pretreating a sample; (3) taking the pre-treated sample as a mold plate, establishing a PCR reaction system by utilizing the primer pair, and carrying out PCR amplification; (4) carrying out agarose gel electrophoresis detection; (5) analyzing the electrophoresis result by utilizing a gel imaging system to obtain a relative strip density value, and quantitatively determining the concentration of the desulfovibro by looking up the standard curve and the linear equation. The quantitative molecular detection method has the advantages that the time consumption is low; the operation is simple; the sensitivity is high; the test charge is low; the detection effect is broad-spectrum; desulfovibro in various samples can be quantitatively detected.

Description

technical field [0001] The present invention relates to the qualitative or quantitative detection of sulfate-reducing bacteria, in particular to a specific primer pair for detecting sulfate-reducing bacteria molecules, a detection method and a detection kit for quantitatively detecting sulfate-reducing bacteria using the primer pair, belonging to sulfate The field of detection of reducing bacteria. Background technique [0002] Sulfate-reducing bacteria were first discovered by Beijerinck in 1895 and have a history of one hundred years. According to incomplete statistics, there are 59 genera and nearly 100 species of sulfate-reducing bacteria, and D. desulfuricans (Desulfuricans) is the most common active bacteria. [0003] Sulfate-reducing bacteria generally grow in soil, river water, and sea water, and it is also found that such bacteria survive in clay soil 71 meters underground and in the seabed at a water depth of 3,000 meters. Identification characteristics: unicellul...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2545/114C12Q2531/113
Inventor 王小通华晶姜利滨郑文涛
Owner 北京世纪金道石油技术开发有限公司
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