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Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of ustilaginoidea virens and application of primer composition

A technology of rice smut and primer composition, applied in the direction of microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problems of no relevant reports, etc., and achieve the effects of improving detection efficiency, shortening detection time, and strong specificity

Inactive Publication Date: 2018-07-24
PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the advantages of fast and sensitive, LAMP technology has been applied in the rapid detection of common pests and diseases of various flowers, plants, and crops, which has greatly improved the identification efficiency and accuracy of plant diseases and insect pests. It is convenient to take measures to control its harm, but there is no relevant report in the detection of rice false smut pathogen

Method used

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  • Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of ustilaginoidea virens and application of primer composition
  • Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of ustilaginoidea virens and application of primer composition
  • Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of ustilaginoidea virens and application of primer composition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The design of embodiment 1 LAMP primer

[0054] Download the UvG-β1 gene (accession number: GU014921.1) gene sequence of rice smut from GenBank for comparison, select a section of gene sequence with high specificity, and use PrimerExplorerV4 software to design LAMP detection primers, including 2 outer primers (F3 and B3), 2 internal primers (FIP and BIP) and two loop primers (Loop F and Loop B), the primer sequences are:

[0055] F3: 5'-GCTCCTGGGATTCTTTGGTG-3';

[0056] B3: 5'-GCTCGATCGGGACAACCA-3';

[0057] FIP: 5'-TTGTCGCGTTGCAAGGACACGGGCATTCGACTCGGAACAG-3';

[0058] BIP: 5'-TTCACTGACATGTGCGCTGACCCGGTTTCGTGGAACGGATT-3';

[0059] Loop F: 5'-CGATTACCGTGCGTGTTGGA-3';

[0060] Loop B: 5'-TCCGCAGTTGAAAATATGG-3'.

[0061] All primers were synthesized at 1OD with ddH 2 O was dissolved and aliquoted. The final concentrations of F3 and B3 were 10 μM, the final concentrations of FIP and BIP were 40 μM, and the final concentrations of Loop F and Loop B were 20 μM, and stor...

Embodiment 2

[0062] Example 2 Establishment of LAMP amplification system

[0063] The reaction system for LAMP amplification was set to 25 μL, and consisted of components with the following concentrations and volumes:

[0064]

[0065] Supplement the volume of the amplification system to 25 μL with sterile double distilled water.

[0066] The reaction condition of LAMP amplification is: 65°C for 60min.

[0067] The chromogenic agent is SYBR Green I.

Embodiment 3

[0068] Example 3 LAMP reaction specificity test

[0069] In this experiment, rice smut bacteria and 17 other common crop pathogens were used as test materials (see Table 1), and the CTAB method was used to extract rice smut bacteria and other common crop pathogen genome DNAs as templates. Referring to the optimized reaction system in Example 2, respectively LAMP amplification was carried out, and the results showed that when the template was rice smut, the color of the reaction product was yellow-green; when the template was other 17 common crop pathogens, the color of the reaction product was orange-red ( figure 1 ). This experiment can well prove that the LAMP reaction of the present invention has excellent specificity for Aspergillus oryzae.

[0070] Table 1 is used for screening the bacterial strain of primer specificity in the present embodiment

[0071] strain

[0072] In the above table: + indicates that the amplified product of LAMP primer has a color chang...

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Abstract

The invention discloses a primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of ustilaginoidea virens and application of the primer composition. The primer composition disclosed by the invention is prepared from the following primers: F3, B3, BIP, FIP, Loop F and Loop B. The primer composition is used for carrying out the LAMP rapid detection on the ustilaginoideavirens and has strong specificity and high sensitivity, so that the primer composition disclosed by the invention can be used for detecting and / or identifying the ustilaginoidea virens; a simple, rapid and low-cost detection technology is provided for scientific researches and production practice and theoretical foundation and technical guidance are also provided for early warning and reasonableapplication of ustilaginoidea oryzae; the primer composition has realistic and profound significances of improving ecological social and economic benefits.

Description

technical field [0001] The invention relates to the field of crop disease detection and identification technology and molecular biology technology, in particular to a primer composition for rapid detection of rice false smut pathogen LAMP and an application thereof. Background technique [0002] Rice false smut (Ustilaginoidea virens) is a worldwide rice panicle disease, also known as false smut, green smut, corn flower disease, blue powdery mildew, commonly known as "high-yield fruit". The disease only occurs in the ears and damages part of the grains. The rice damaged by rice mistletoe shows that the mycelium blocks formed in the grains gradually expand, and the inner and outer glumes split to reveal light yellow lumps, that is, spores, which are later wrapped on both sides of the inner and outer glumes, which are black and green. The first layer of film ruptures, and dark green powder is scattered, that is, the chlamydospores of the pathogen, and some black flat scleroti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 陈雨杨雪谷春艳臧昊昱
Owner PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
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