Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of pomegranate fusarium sulphureum and application of primer composition

A technology of pomegranate dry rot pathogen and primer composition, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., to achieve the effects of shortening detection time, improving efficiency, and strong specificity

Active 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. Measures to control its harm provide convenience, but there is no relevant report in the detection of pomegranate dry rot pathogen

Method used

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  • Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of pomegranate fusarium sulphureum and application of primer composition
  • Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of pomegranate fusarium sulphureum and application of primer composition
  • Primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of pomegranate fusarium sulphureum and application of primer composition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The design of embodiment 1 LAMP primer

[0052] Download the elongation factor 1-alpha gene (accession number: JQ281778) gene sequence of Pomegranate dry rot from GenBank, according to the gene sequence, use PrimerExplorer V4 software to design a kind of LAMP detection primer, including 2 outer primers (F3 and B3) and 2 internal primers (FIP and BIP), the primer sequences are:

[0053] F3: 5'-GGTCACCGTGACTTCATCAA-3';

[0054] B3: 5'-CCTTCCACTCAGCAGTGTC-3';

[0055] FIP: 5'-CACCAGTACCGGAGGCAATGATCATGATCACTGGTACCTCGC-3'; BIP: 5'-GCTGGTATCTCCAAGGATGGCCACGATGAGCTGCTTGACAC-3'.

[0056] All primers were synthesized at 1OD with ddH 2 After O was dissolved, it was aliquoted, and the final concentration of F3 and B3 was 10 μM, and the final concentration of FIP and BIP was 40 μM, and stored at 4°C for future use.

Embodiment 2

[0057] The establishment of embodiment 2 LAMP amplification system,

[0058] Use two pairs of specific primers to carry out constant temperature LAMP amplification of the genomic DNA of the fungus to be identified. The total volume of the LAMP reaction is 20 μL. The reaction system is:

[0059]

[0060]

[0061] Supplement the total volume of the amplification system to 20 μL with sterile double distilled water.

[0062] The reaction condition of LAMP amplification is: 65°C for 60min.

[0063] The chromogenic agent is SYBR Green I.

Embodiment 3

[0064] Example 3 Detection of LAMP Reaction Specificity of Pomegranate Dry Rot Pathogen

[0065] In this experiment, the pomegranate dry rot fungus and 22 other common crop pathogens were used as test materials (see Table 1). The genomic DNA of the pathogenic bacteria of crops was used as a template, and the reaction system established in Example 2 was used to perform LAMP amplification respectively. When the template was pomegranate dry rot bacteria, the electrophoresis pattern formed ladder-like bands; when the template was other common pathogenic bacteria of crops, there was no bands, indicating that the LAMP reaction has good specificity (see figure 1 ).

[0066] Table 1 is used for screening the bacterial strain of primer specificity in the present embodiment

[0067]

[0068]

[0069] In the above table: + indicates that there are LAMP primer amplification bands and color changes; - indicates no amplification bands and color changes.

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Abstract

The invention discloses a primer composition for LAMP (loop-mediated isothermal amplification) rapid detection of pomegranate fusarium sulphureum and application of the primer composition. The primercomposition disclosed by the invention is prepared from the following primers: F3, B3, BIP and FIP. The primer composition is used for carrying out the LAMP rapid detection on the pomegranate fusariumsulphureum 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 pomegranate fusarium sulphureum;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 reasonable application of the pomegranate fusarium sulphureum; the primer composition has realistic and profound significances of increasing 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 pomegranate dry rot pathogen LAMP and an application thereof. Background technique [0002] The causative bacteria of pomegranate dry rot belong to the subphylum Deuteromycota, and the fungus of the genus Ascospora. The pycnidia are spherical or oblate, and the conidia are diamond-shaped, dark yellow, and often stick together with mucus. The hyphae on the stiff fruit produce new spores around mid-April of the following year and become the source of transmission of the disease. It has occurred in many provinces of our country, seriously affecting the pomegranate production and food security in our country. In order to prevent the continuous expansion of the spread of pomegranate dry rot, it is particularly important to detect and identify it quickly and accurately. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107C12Q2565/125
Inventor 杨雪陈雨谷春艳臧昊昱
Owner PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
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