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Method for identifying Trichoderma fungi by loop-mediated isothermal amplification and primers thereof

A ring-mediated isotherm, Trichoderma technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of expensive instrument operation experience and dependence, and achieve efficiency improvement and specificity Highly specific and highly specific effects

Pending Publication Date: 2020-04-10
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many molecular detection techniques for the rapid identification of Trichoderma species diseases, most of them rely on expensive instruments and rich operating experience

Method used

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  • Method for identifying Trichoderma fungi by loop-mediated isothermal amplification and primers thereof
  • Method for identifying Trichoderma fungi by loop-mediated isothermal amplification and primers thereof

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Embodiment 1

[0043] Example 1: A method and primers for identifying Trichoderma fungi by loop-mediated isothermal amplification

[0044] Design of primers: During the identification of Agaricus bisporus disease, a Trichoderma viridans was collected T. virens strain, and its ITS sequence was determined (GenBank No. MK552404). Based on this, primers were designed upstream outer primer F3, downstream outer primer B3, forward inner primer FIP, and reverse outer primer BIP. This application designed loop primers LB (B-Loop Primers), which can promote LAMP reactions. Among them, both FIP and BIP are composed of two sequences. Due to the presence of "-" in the designed primer, we changed the position of the short line into "TTTT" flexible linker when synthesizing the primer later, and the final primer uses a solid phase Synthesized by phosphoramidite triester method, and the synthesis of primers was completed by Shanghai Jierui Bioengineering Co., Ltd.

[0045] The genomic DNA of 5 Trichoderma...

Embodiment 2

[0095] Example 2: A method and primers for identifying Trichoderma fungi by loop-mediated isothermal amplification

[0096] A primer for identifying Trichoderma fungi by loop-mediated isothermal amplification method, including upstream outer primer F3, downstream outer primer B3, forward inner primer FIP, reverse outer primer BIP and loop primer LB, the sequence of which is:

[0097] Upstream outer primer F3: 5'-CAGTATTCTGGCGGGCATG-3';

[0098] Downstream outer primer B3: 5'-ACCTGATCCGAGGTCAACAT-3';

[0099] Forward internal primer FIP: 5'-TAAAGGGCCGATCCCCAACGTTTTGTCCGAGCGTCATTTTCAACC-3';

[0100] Reverse outer primer BIP: 5'-CAGCCTCTCCTGCGCAGTAGTTTTGGGTGTTTAACGGCTGTGG-3';

[0101] Loop primer LB: 5'-TTTGCACACTCGCATCGGGA-3'.

[0102] In order to achieve the above object and other related objects, the technical solution provided by the present invention is: a method for identifying Trichoderma fungi by a ring-mediated isothermal amplification method, comprising the following...

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Abstract

The invention relates to a method for identifying Trichoderma fungi by loop-mediated isothermal amplification and primers thereof. The primers comprise an upstream outer primer F3, a downstream outerprimer B3, a forward inner primer FIP, a reverse inner primer BIP and a loop primer LB. LAMP reaction conditions: adding all reagents of the LAMP reaction system into a reaction vessel, and then adding 0.25 uL of SYBR Green I nucleic acid gel dye; and then placing the reaction vessel at the temperature of 65 DEG C and incubating for 60 min. Determination of reaction results: the result of a non-specific reaction is orange with no fluorescence; and the result of a specific reaction is yellow-green with fluorescence. Compared with a PCR method, the method of the invention has higher specificityfor identifying Trichoderma fungi; meanwhile, isothermal amplification is adopted without thermal cycling steps such as denaturation, degeneration and renaturation, and the efficiency is remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and relates to a method and primers for identifying Trichoderma fungi by a loop-mediated isothermal amplification method. Background technique [0002] Trichoderma ( Trichoderma ) disease is the most easily infected and transmitted disease in the cultivation process of Agaricus bisporus. It is mainly distributed in the form of conidia in the cultivation materials of Agaricus bisporus and the surrounding environment of the mushroom field, and will join the Agaricus bisporus mushroom bed that is growing or fruiting with the wind, insects, people, etc.; it grows fast and lives It is strong and can produce a large amount of white hyphae within 2-4 days; its hyphae can infect the entire mushroom bed, preventing the mycelia of Agaricus bisporus from growing, infecting the fruiting bodies of Agaricus bisporus, and improving the quality of Agaricus bisporus Fall or die. Finally, piles of green ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2537/1376C12Q2563/107
Inventor 宋晓霞黄建春陈明杰王倩隽加香陈辉张津京
Owner SHANGHAI ACAD OF AGRI SCI
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