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

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

Inactive Publication Date: 2008-03-05
HARBIN INST OF TECH
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AI Technical Summary

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 counting result. A rapid quantitative detection method for direct doubling dilution PCR of sulfate-reducing bacteria that effectively shortens the counting time, truly reflects the actual production situation, and reduces testing costs. It uses the doubly-diluted sulfate-reducing bacteria as a template Direct PCR amplification detection

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

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specific Embodiment approach 1

[0005] Specific embodiment one: present embodiment is a kind of sulfate-reducing bacteria direct doubling dilution PCR rapid quantitative detection method, and it comprises the following steps successively: (1).Preparation thalline pretreatment buffer; (2).Thalline preparation Preparation; (3). Doubling dilution; (4). Carry out PCR operation on ice; (5). PCR amplification on the amplification instrument; (6). 1.0% agarose electrophoresis detection; (7) .Result observation, look-up table counting; the PCR reaction system in the process of "(4). Performing PCR operation on ice" is 20 μl: including: Buffer (10×) 2 μl, 0.3mmol L -1 dNTP 2μl, the concentration is 0.1μmol L -1 The forward primer SRBF 1μl, the concentration is 0.1μmol L -1 Add 1 μl of reverse primer SRBR, 0.3 U of rTaq enzyme, add 11.7 μl of deionized water to the rest, and then add 2 μl of sample.

specific Embodiment approach 2

[0006] Specific embodiment two: the detailed operation process of this embodiment is as follows:

[0007] (1). Preparation of bacterial cell pretreatment buffer: the mother liquor composition of "bacterial cell pretreatment buffer" is: 70gNaCl, 2g KCl, 12g NaCl 2 HPO 4 , 2g KH 2 PO 4 , 1‰SDS; dilute the mother liquor 10 times to make the bacteria pretreatment buffer; control the pH of the working solution to 7.5, and sterilize it at 121°C for 15-30min.

[0008] (2). The preparation of the bacteria: including the following steps in turn: a. The preparation of the bacteria is completed under the ultra-clean workbench. It is necessary to sterilize the ultra-clean workbench with ultraviolet rays for 20-40 minutes, and then use high-purity nitrogen Take 1mL of the oilfield sewage water sample retrieved by the anaerobic tube, or the sludge of the bioreactor, and inject it into a sterilized 1.5mL centrifuge tube; b. fully shake the oilfield sewage water sample on the shaker For 3...

specific Embodiment approach 3

[0014] Specific implementation mode three: In this implementation mode, the oilfield sewage retrieved from the anaerobic tube containing high-purity nitrogen is used as the test sample, and the five-tube parallel method is used for the PCR operation on ice, with 5 samples for each gradient. 1.0% agarose electrophoresis detection, the detection results are shown in Figure 1 and Figure 2. Observing the results, looking up the table and counting.

[0015] The specific look-up table calculation method is: select the last three serial dilution gradients (10 x 、10 x+1 、10 x+2 ), arrange the number of tubes with bacterial DNA bands in descending order of the dilution gradient into a three-digit number called the band approximation (abc). Among them, the first dilution gradient (10 x ) is the dilution gradient in which the bacterial DNA bands appear in the last 5 test tubes, if the third dilution gradient (10 x+2 ) and the further down gradient dilution tubes still have cell DNA ...

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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) 1.0% 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/25C12Q1/04C12Q1/68
Inventor 马放魏利
Owner HARBIN INST OF TECH
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