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Kit and method for rapidly detecting archaea

A kit and rapid technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low frequency of use, high sequencing error rate, long detection cycle, etc., to achieve specificity Strong, low experimental cost, easy to operate

Pending Publication Date: 2021-04-13
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Real-time fluorescent quantitative PCR and high-throughput sequencing are now mostly used to explore the abundance, diversity, composition and structure of archaeal communities in different environments. Real-time fluorescent quantitative PCR detection of archaea is subject to: the required reagents are expensive and the number of experiments used is less , it is impossible to detect and identify archaea in batches
Although gene sequencing is easy to operate, the problem is that the cost of sequencing is high, the error rate of sequencing is high, and it relies on large-scale scientific research instruments and professional bioinformatics analysts, and the detection cycle is relatively long

Method used

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  • Kit and method for rapidly detecting archaea
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  • Kit and method for rapidly detecting archaea

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1. A primer set and DNA fragments for rapid detection of archaea by chemical synthesis.

[0037] Among them, the member sequences of the primer set are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; the sequences of all DNA fragments are SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0038] 2. The chemically synthesized DNA fragments were amplified by PCR using the primer pair SEQ ID NO: 1 (upstream primer) / SEQ ID NO: 3 (downstream primer), and the amplified product was used as a standard for the sample to be tested.

[0039] 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 DNA ( Chemically synthesized DNA fragments): 1 μL, ddH20: 37 μL;

[0040] The PCR reaction program was: 94°C for 5min; 30 cycles of 94°C for 30s, 45-55°C for...

Embodiment 2

[0044] The method for detecting the activated sludge archaea in the aeration tank of a sewage treatment plant: comprises the following steps:

[0045] 1. Extract the total DNA of the microbial genome from the activated sludge in the aeration tank

[0046] (1) Dilute and dissolve the activated sludge with ultrapure water, filter it with a 0.22 μm filter membrane, put the filter membrane with microorganisms into an EP tube, wash it twice with STE buffer, and then centrifuge at 12000 rpm for 1 minute.

[0047] (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.

[0048] (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.

[0049] (4) Take it out after completion, cool to room temperature, and add 70 μl of NaCl.

[0050] (5) Add 450 μl DNA extraction phenol reagent, mix for 10 minutes, centrifuge at 12000 r...

Embodiment 3

[0066] A method for detecting archaea in sediments of rural biogas digesters: comprising the following steps:

[0067] 1. Extraction of microbial genome total DNA in biogas digester sediment

[0068] 1.1 DNA extraction

[0069] (1) Dilute the solid-liquid sample in the biogas digester with ultrapure water, and then filter it with a 0.22 μm filter membrane, put the filter membrane with archaeal microorganisms into the EP tube, wash it twice with STE buffer, and then Centrifuge at 12000 rpm for 1 min.

[0070] (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.

[0071] (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.

[0072] (4) Take it out after completion, cool to room temperature, and add 70 μl of NaCl.

[0073] (5) Add 450 μl DNA extraction phenol reagent, mix for 10 minutes, centrifuge at 120...

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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 archaea. According to the kit and the method for rapidly detecting archaea, the problems existing in abundance, diversity, composition and structure of archaea communities under different environments in real-time fluorescent quantitative PCR and high-throughput sequencing at present are solved, and the kit for quickly detecting archaea comprises a primer group, a DNA fragment, a PCR buffer solution and an STE buffer solution; in addition, the DGGE technology is used for rapidly detecting the archaea, the experiment cost is low, operation is easy and convenient, and meanwhile the method has the advantages of being high in specificity, easy to popularize in a laboratory, independent of large experiment equipment and the like; and the method is an efficient, rapid, economical and convenient detecting method.

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 archaea. Background technique [0002] Archaea is the third form of life, which contains the early information of life evolution. Archaea are widely distributed in various environments on the earth (including extreme natural environments), with extremely rich genetic and metabolic diversity, and are important drivers of elemental biogeochemical cycles. At the same time, archaea and their metabolites also show unique potential for biotechnology development. [0003] Methanogenic bacteria are a very special group of organisms, belonging to archaea. This type of bacteria has a special energy-producing metabolic function. It can use organic or inorganic substances as substrates and convert them into methane und...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12Q1/04
CPCC12Q1/689C12Q1/686C12Q2565/125C12Q2563/173
Inventor 杨秀清徐现
Owner SHANXI UNIV
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