Kit and method for rapidly detecting coal geological environment bacteria

A geological environment, kit technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Limited capacity, etc., to achieve the effect of fast experiment speed, reliable results and low price

Pending Publication Date: 2021-04-09
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, although high-throughput sequencing has high accuracy, the cycle for microbial diversity analysis is long, time-consuming and expensive, and the cost is high
The library capacity of clone library construction is limited, and often cannot accurately reflect the composition of the microbial community structure in the original sample

Method used

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  • Kit and method for rapidly detecting coal geological environment bacteria
  • Kit and method for rapidly detecting coal geological environment bacteria
  • Kit and method for rapidly detecting coal geological environment bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. A primer set and DNA fragments for the rapid detection of coal geological environment bacteria by chemical synthesis.

[0035] The primer set includes: detection primers for amplifying coal geological environment bacteria, the sequences of which are respectively shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;

[0036] The DNA fragments are bacterial DNA detection fragments in the coal geological environment, and their sequences are respectively SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9. The sequence shown in SEQ ID NO:10.

[0037] 2. Perform PCR amplification on the chemically synthesized DNA fragments using the primer pair SEQ ID NO: 1 / SEQ ID NO: 3, and the amplified product is used as the standard of the sample to be tested.

[0038] The PCR reaction system is: 10*EasyTaq Buffer: 5 μL, dNTPs (2,5 mM): 4 μL, upstream primer (10 μmol / L): 1 μL, downstream primer (10 μmol / L): 1 μL, EasyTaq DNA Polymerase: 1 μL, template ...

Embodiment 2

[0043] The method for detecting bacteria in a coal mine effluent sample: comprises the following steps:

[0044] 1. Extract the total DNA of the water sample microbial genome

[0045] (1) Suction filter the collected water sample with a 0.22 μm filter membrane, put the filter membrane with microorganisms into an EP tube, wash twice with STE buffer, and then centrifuge at 12000 rpm for 1 minute.

[0046] (2) Add 300 μl of STE buffer solution and 30 μl of lysozyme, wash and mix with a pipette, and place in a constant temperature water bath at 37° C. for 3.5 hours.

[0047](3) Add 35 μl of SDS and 5 μl of Proteinase K, and keep in a constant temperature water bath at 55° C. for 2.5 hours.

[0048] (4) Take it out after completion, cool to room temperature, and add 70 μl of NaCl.

[0049] (5) Add 450 μl DNA extraction phenol reagent, mix for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and take the supernatant.

[0050] (6) Add an equal volume of phenol-chloroform-isoamy...

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Abstract

The invention belongs to the field of microbial molecular ecology detection, and particularly relates to a kit and a method for rapidly detecting coal geological environment bacteria. Aiming at the problem of limitation of environment bacteria detection in the prior art, the invention provides the kit for rapidly detecting the coal geological environment bacteria. The kit comprises a primer group and a DNA fragment for rapidly detecting the coal geological environment bacteria, a PCR buffer solution and an STE buffer solution. In addition, the geological environment bacteria are rapidly detected by using a DGGE technology, the result is reliable, the experiment speed is high, the specificity is good, the sensitivity is high, and a plurality of samples can be analyzed at the same time. The price of experimental consumables is low, and the experimental technology is easy to popularize and simple to operate. Compared with a general detection method, the detection method is efficient, rapid, economical and convenient.

Description

technical field [0001] The invention belongs to the field of microbial molecular ecology detection, relates to gel electrophoresis (DGGE) technology related to the detection of environmental microbial communities, in particular to a kit and method for rapid detection of bacteria in coal geological environments. Background technique [0002] Coal geological microorganisms refer to the microorganisms detected in coal seam output water samples or coal samples, or the above two microorganisms detected after enrichment and culture. They are mainly divided into two categories: bacteria and archaea, which are mainly involved in the process of degrading coal to produce methane middle. Therefore, analyzing the diversity of gas-producing microbial communities helps to grasp the types of functional microorganisms, and lays a theoretical foundation for the regulation of microbial gas formation and the revealing of microbial gas formation mechanisms. However, through conventional method...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12Q1/04
CPCC12Q1/689C12Q1/686C12Q2600/166C12Q2565/125C12Q2563/173
Inventor 杨秀清丁超
Owner SHANXI UNIV
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