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Quick-speed PCR quantitative determination method of sulfate-reducing bacteria direct dilution by multiple proportions

A quantitative detection method and multiple dilution technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of long detection cycle, inability to guide production in real time, and high detection cost

Inactive Publication Date: 2006-05-10
HARBIN INST OF TECH
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  • Claims
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Problems solved by technology

[0003] Aiming at the problems in the existing detection of SRB bacteria in oilfield water quality that the detection period is long, the number of SRB bacteria in the water cannot be truly and comprehensively reflected, the production cannot be guided immediately, and the detection cost is high, so as to provide an accurate and reliable

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  • Quick-speed PCR quantitative determination method of sulfate-reducing bacteria direct dilution by multiple proportions
  • Quick-speed PCR quantitative determination method of sulfate-reducing bacteria direct dilution by multiple proportions

Examples

Experimental program
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Example Embodiment

[0005] Specific embodiment 1: This embodiment is a method for rapid quantitative detection of sulfate-reducing bacteria by direct doubling dilution PCR, which sequentially includes the following steps: (1). Preparation of cell pretreatment buffer; (2). Bacterial cell Preparation; (3). Time ratio dilution; (4). PCR operation on ice; (5). PCR amplification on a thermal cycler; (6). 1.0% agarose electrophoresis detection; (7) Observe the results, check the table and count; the PCR reaction system in the process of "(4). Perform PCR operation on ice" is 20μl: including: Buffer (10×) 2μl, 0.3mmol·L -1 D NTP 2μl, concentration is 0.1μmol·L -1 The forward primer SRBF 1μl, the concentration is 0.1μmol·L -1 1μl of reverse primer SRBR, 0.3U rTaq enzyme, 11.7μl of deionized water for the rest, and then 2μl of sample.

Example Embodiment

[0006] Specific implementation manner 2: The detailed operation process of this implementation manner is as follows:

[0007] (1). Preparation of cell pretreatment buffer: the mother liquor components of "Bacterial cell pretreatment buffer" are: 70g NaCi, 2g Kci, 12g Na 2 HPO 4 , 2g KH 2 PO 4 , 1‰ SDS; Dilute the mother liquor 10 times to become the pretreatment buffer of the bacteria; control the pH of the working solution to 7.5 and sterilize it at 121°C for 15-30 minutes.

[0008] (2). Preparation of bacterial cells: The following steps are included in sequence: a. The preparation of bacterial cells is completed under an ultra-clean workbench, which needs to be sterilized by ultraviolet light for 20-40 minutes, and then used with high-purity nitrogen Take 1mL of the oilfield sewage water sample retrieved by the anaerobic tube or bioreactor sludge and inject 1mL into the sterilized 1.5mL centrifuge tube; b. Fully shake the oilfield sewage water sample on the shaker 3~6min, fully...

Example Embodiment

[0014] Specific embodiment 3: In this embodiment, the oilfield sewage retrieved by the anaerobic tube containing high-purity nitrogen is used as the test sample, and the five-tube parallel method is used to perform the PCR operation on ice, with 5 samples for each gradient. 1.0% agarose electrophoresis detection, the detection result is as follows figure 1 , figure 2 Shown. Observe the results, check the meter and count.

[0015] The specific look-up table calculation method is: select the last three serial dilution gradients (10 x , 10 x+1 , 10 x+2 ), the number of tubes with bacterial DNA bands is arranged in the order of the dilution gradient from small to large into a three-digit number called the band approximate value (abc). Among them, the first dilution gradient (10 x ) Is the last dilution gradient with bacterial DNA bands in the last 5 test tubes. If the third dilution gradient (10 x+2 ) There are still bacterial DNA bands appearing in the next gradient dilution tube, t...

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Abstract

This invention discloses to a sulfate reducing bacterium timely diluting PCR rapid quantitative determination method, which relates to the sulfate reducing bacterium (SRB) quantitative determination method. This invention comprises the following steps: (1) preparing 'thalli formal dispose cushion succus'; (2)preparing the thalli;(3)timely diluting; (4) making the PCR operation on the ice; (5) PCR expanding on the expanding device; (6) 1.0úÑ agarose tiselius detectiong the expanded production;(7) Observing result, recording number. The result of this method is accurate, which can shorten the account time effectively and reflect the physical production condition actually.

Description

technical field [0001] The invention relates to a rapid and quantitative detection method for sulfate-reducing bacteria (SRB) in the water treatment process of conventional sewage, poly-containing sewage and surface technology in an oil field water system. Background technique [0002] At present, the counting of SRB bacteria in the oilfield system mainly follows the Chinese oil and gas industry standard, "Bacterial Analysis Method for Oilfield Injection Water - Extinct Dilution Method (MPN)" (Ministerial Standard), SY-T0532-93. The problem with the above-mentioned method is that the detection takes 14 days. Due to the long detection cycle, it cannot effectively and immediately guide the production practice. In the actual operation process, some strict anaerobic SRB bacteria cannot survive, resulting in SRB The counting result of bacteria is inaccurate, cannot truly reflect the actual production situation, and the detection cost is high; and because the existing detection me...

Claims

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Application Information

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IPC IPC(8): C12Q1/25C12Q1/04C12Q1/68
Inventor 马放魏利
Owner HARBIN INST OF TECH
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