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

A real-time fluorescence quantitative and soil technology, applied in the direction of microbial-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of not being able to obtain reliable information on the number of fungi, not being able to characterize the overall number of soil fungi, and derailing experimental equipment and other problems, to achieve the effect of eliminating the artificial cultivation or leaching extraction steps, accurate and reliable results, and high test sensitivity

Pending Publication Date: 2020-04-24
NANJING FORESTRY UNIV
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Problems solved by technology

However, the methods for the determination of the number of soil fungi in my country use the dilution plate method, agar film method, ergosterol conversion method, and chitin conversion method. However, there are many problems such as extensive and cumbersome methods, derailment from international advanced methods, and backward experimental equipment.
Taking the dilution plate method as an example, since the total number of known fungal species in the world is as high as 5.1 million, far exceeding the original estimate of 1.5 million species, but only 80,000 of them can be cultured artificially, the plate culture method has great limitations , can not obtain reliable information on the number of fungi; the ergosterol and chitin conversion method is based on the common and unique substances of fungi to estimate the number of soil fungi. Although the method is simple and easy to implement, it has poor specificity and low data accuracy; and Using real-time fluorescent quantitative PCR to detect a certain type of fungi with special physiological functions, such as invention patent 201610473539.8, discloses a method for detecting the number of arbuscular mycorrhizal fungi in wheat rhizosphere soil based on real-time fluorescent quantitative PCR. Although this method has high sensitivity, it cannot Characterize the overall number of soil fungi

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

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: establish the method based on real-time fluorescence quantitative PCR to detect the amount of soil fungus

[0033]Fluorescence designed for the 18S ribosomal rRNA conserved sequence of common soil fungi (Phlebiopsis gigantea, Trichoderma, Heterobasidion parviporum, and Stereum sanguinolentum) Quantitative PCR (qPCR) general primers are as follows:

[0034] Sequence of upstream primer FF390: 5'-CGATAACGAACGAGACCT-3';

[0035] Sequence of downstream primer FR1: 5'-AICCATTCAATCGGTAIT-3', wherein I is inosine base.

[0036] Standard curve template DNA serial dilution concentration: using the known genome base number (40.92Mb) and quality (4.48432×10 -5 ng) and its target-specific fragment copy number (5 / genome), calculate the target-specific fragment copy concentration (copy / μL) in the standard curve template DNA mother solution; take an appropriate amount of mother solution and add sterilized double distilled water according to the dilution formula Adjust ...

Embodiment 2

[0047] The soil used in this example was collected from the southwest foot of Zijin Mountain (32°04′N, 118°50′E) in Nanjing City, Jiangsu Province, and the mixed forest of sweetgum and black pine. Weakly acidic. Remove the litter layer about 1m away from the main trunk of the tree, and use a sterilized 50mL centrifuge tube to take the surface soil (0-10cm). Treatment 1 is the soil of liquidambar pure forest, and treatment 2 is the soil of liquidambar black pine mixed forest. Each treatment is performed 3 times Repeat, use the above method to detect the number of soil fungi.

[0048] The specific operation steps are as follows:

[0049] 1) Pass the soil sample through a 2mm sieve, weigh about 0.3g of fresh soil, use the OMEGA Soil DNA Extraction Kit to extract the total DNA of the soil, dissolve it in sterilized double distilled water, measure the DNA concentration, and place it in a -20°C refrigerator for later use.

[0050] 2) The hyphae of the template Phlebia radiata FBCC...

Embodiment 3

[0062] The soil used in this example was collected from the southern seedling base (21°27'N, 110°11'E) of Zhanjiang City, Guangdong Province, 4-year-old and 9-year-old red eucalyptus 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 red eucalyptus plantation, and treatment 2 is the soil of the 9-year-old red eucalyptus plantation. 3 repetitions. Concrete operation step is with embodiment 2, draws this standard curve, y=-3.4834x+41.119; Wherein R 2 =0.9904, amplification efficiency E=93.68%.

[0063] The test results are as follows:

[0064]

[0065]

[0066] The data in the table also shows the high precision and reproducibility of the results of this analysis method, and also shows that different forest ages have a significant impact on the number of soil fungi.

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Abstract

The invention discloses a method for detecting the number of soil fungi based on real-time fluorescent quantitative PCR, and belongs to the field of detection of the number of soil fungi. The method disclosed by the invention mainly comprises the following steps: weighing sieved fresh soil, extracting total DNA of the soil by using A test kit, and uniformly diluting to a certain concentration; extracting total DNA (deoxyribonucleic acid) from mycelia of moderately growing template Phlebia radiata by using a CTAB (cetyltrimethyl ammonium bromide) method to prepare series template DNA solutionsof a standard curve; constructing a qPCR reaction system by using the standard curve or the to-be-detected soil template DNA solution, wherein the qPCR reaction system comprises a universal qPCR primer designed according to the conserved sequence of the rRNA of fungus 18S ribosome; carrying out qPCR reaction by using an ABI7500 real-time fluorescent quantitative PCR instrument; drawing the standard curve; and calculating the number of fungi in each gram of dry soil. The method for detecting the number of soil fungi provided by the invention is safe and simple to operate, good in primer sequence specificity, high in sensitivity, accurate and reliable in result and good in reproducibility.

Description

technical field [0001] The invention belongs to the field of soil fungal quantity detection, and more specifically relates to a method for detecting soil fungal quantity based on real-time fluorescent quantitative PCR (qPCR). Background technique [0002] Fungi play an important role in agricultural and forest soil ecosystems. The number of fungi per gram of soil reaches tens of millions, and the number of fungi in rhizosphere soil reaches 100 times. Studies have shown that fungi are an important link between soil and plants, and are closely related to plant growth. On the one hand, fungi are the main microorganisms that degrade organic matter, and the degrading enzymes they secrete are an important guarantee for soil nutrient conversion and energy flow. On the other hand, fungi secrete plant growth-promoting or harmful substances to enhance plant disease resistance or induce plant disease. At the same time, fungi are also the main components of soil microorganisms. The glo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/06C12R1/645
CPCC12Q1/6851C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 刘兵曲朝磊马阳徐杰刘怡杨艺红孙辉
Owner NANJING FORESTRY UNIV
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