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Method for detecting number of soil bacteria based on real-time fluorescent quantitative PCR

A real-time fluorescent quantitative and bacterial technology, applied in the biological field, can solve the problems such as the inability to obtain reliable information on the number of bacteria by the dilution plate method, the inappropriate promotion of high-throughput sequencing, and the low accuracy of observation and counting. Difficulty, easy to operate and easy to learn

Pending Publication Date: 2020-04-24
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] For the deficiencies in the prior art, the object of the present invention is to provide a kind of method based on real-time fluorescent quantitative PCR detection soil bacterial quantity, can not obtain the reliable information of bacterial quantity to solve dilution plate method; Due to the influence of dye fluorescence quenching factors, the accuracy of observation and counting is not high; high-throughput sequencing is not suitable for promotion, and quantitative detection of qPCR requires the preparation of culture medium, preparation of competent cells and extraction of plasmids, and the detection steps are cumbersome.

Method used

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  • Method for detecting number of soil bacteria based on real-time fluorescent quantitative PCR
  • Method for detecting number of soil bacteria based on real-time fluorescent quantitative PCR
  • Method for detecting number of soil bacteria based on real-time fluorescent quantitative PCR

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Embodiment 1

[0027] The soil used in this example was collected from fertilized bamboo forests and unfertilized bamboo forests at the northern foot of Zijin Mountain (32°01'N, 118°49'E) in Nanjing City, Jiangsu Province. The soil type is yellow brown soil and weakly acidic. Remove the litter layer about 0.5m away from the trunk of the tree, and use a sterilized 50mL centrifuge tube to take the surface soil (0-10cm). Treatment 1 is the fertilized bamboo forest soil, and treatment 2 is the non-fertilized bamboo forest soil. Each treatment is repeated 3 times. Methods To detect the number of soil bacteria.

[0028] The specific operation steps are as follows:

[0029] 1) Pass the soil sample through a 2mm sieve to remove debris such as stones, tree roots and fallen leaves, weigh about 0.3g of fresh soil, and use the OMEGA Soil DNA Extraction Kit (OMEGA E.Z.N.A. Soil DNA Extraction Kit type is 50 times / box , Cat. No. D5625-01) to extract total DNA from soil, dissolved in ddH 2 In O, the DNA ...

Embodiment 2

[0044] The soil used in this example was collected from the South Seedling Base (21°27'N, 110°11'E) of Zhanjiang City, Guangdong Province, 4-year-old and 14-year-old Eucalyptus rugosa plantations. The soil type is brick red soil, which is weakly acidic. Remove the litter layer about 1m away from the main trunk of the tree, and take the surface soil (0-10cm) with a sterilized 50mL centrifuge tube. Treatment 1 is the soil of the 4-year-old Eucalyptus ivory plantation, and Treatment 2 is the soil of the 14-year-old Eucalyptus ivory plantation. 3 repetitions. Concrete operation step is with embodiment 1, draws this standard curve, y=-3.4484x+36.632; Wherein R 2 =0.9986, amplification efficiency E=94.98%. The results are shown in Table 2:

[0045] Table 2 Detection of soil bacteria in 4-year-old and 14-year-old Eucalyptus scutellaria plantations

[0046]

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Abstract

The invention discloses a method for detecting the number of soil bacteria based on real-time fluorescent quantitative PCR, and belongs to the technical field of biology. The method comprises the following steps: 1) extracting soil sample total DNA and adjusting the total DNA to a preset concentration to prepare a to-be-detected soil sample template DNA solution; 2) extracting the total DNA of template escherichia coli, measuring the DNA concentration, and preparing a standard curve template DNA solution; 3) carrying out real-time fluorescent quantitative PCR amplification on the to-be-detected soil sample template DNA solution and the standard curve template DNA solution; 4) drawing a standard curve; and 5) calculating the number of bacteria per gram of dry soil. According to the method,the steps of adding an internal standard strain for fluoroscopy and the like are omitted, the operation difficulty is greatly reduced, connection, conversion and qPCR amplification are not needed, operation is easy, convenient and easy to learn, and the target fragment is good in specificity, high in data accuracy and good in reproducibility.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting the number of soil bacteria based on real-time fluorescent quantitative PCR. Background technique [0002] Soil bacteria are not only the core species in the soil ecosystem, but also the most abundant and diverse microbial taxonomy, accounting for about 90% of the total number of microorganisms. Soil bacteria regulate the cycling of elements such as carbon and nitrogen and the mineralization of organic matter, which play a very important role in soil biochemical processes and energy flow. Among them, nitrogen elements (such as nitrogen fixation, nitrification and denitrification), phosphorus elements (phosphorus solution) and potassium elements (potassium solution) can only be completed by soil bacteria. Since the number of soil bacteria is an important indicator of the size and function of microbial communities, more and more studies have paid att...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2545/114C12Q2537/165C12Q2563/107
Inventor 刘兵马阳曲朝磊徐杰耿莉孙辉
Owner NANJING FORESTRY UNIV
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