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RPA primer for detecting gibberella fujikuroi of rice bakanae disease and detection method

A technology for rice bakanae disease and Gibberella Fujikura, which is applied in the detection field of RPA primers and detection of Gibberella Fujikura, the pathogen of rice bakanae disease, and can solve the problems that have not yet been reported on the detection of Gibberella Fujikura

Inactive Publication Date: 2020-07-07
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

[0004] At present, RPA technology has been widely used in the rapid detection of viruses, bacteria, mycoplasma and parasites, but there is no report on the detection of Gibberella fujikura

Method used

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  • RPA primer for detecting gibberella fujikuroi of rice bakanae disease and detection method
  • RPA primer for detecting gibberella fujikuroi of rice bakanae disease and detection method
  • RPA primer for detecting gibberella fujikuroi of rice bakanae disease and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Screening of RPA Primers of Gibberella Fujikura, the Pathogen of Rice Bakanae Disease

[0039] Taking the sequence of Gibberella fujikura TEF-1 gene (GeneBank No.GQ848524.1) as the target, according to the design principles of RPA primers, the primers used in the RPA kit (TwistAmp nfo) were designed and screened to obtain high amplification efficiency, Primer pairs with the best sensitivity and specificity. The optimal primer (being the forward primer of RPA primer and the reverse primer) sequence obtained by screening is as shown in SEQ ID No.1 and SEQ ID No.2:

[0040] Forward primer Ff-RPA-F (SEQ ID No.1): 5'-GCTTATCTGTCATCGTGATCCTGACCAAGATC-3',

[0041] Reverse primer Ff-RPA-R (SEQ ID No.2): 5'-TGGACAGGAAAGGGCAAAACGCGCCCATCACTCG-3':

[0042] The RPA amplified product of above-mentioned primer (obtained by the obtaining method of Gibberella fujikura RPA amplified product) carries out the electrophoresis detection of agarose gel, figure 1 The mark "2" in refers to ...

Embodiment 2

[0053] Specific Detection of RPA Primers of Gibberella Fujikura, the Pathogen of Rice Bakanae Disease

[0054] Refer to the method for obtaining the RPA amplification product of rice bakanae disease pathogen Gibberella Fujikura, wherein the primers are forward primer Ff-RPA-F and reverse primer Ff-RPA-R, wherein the samples are Gibberella Fujikura, cereal Fusarium, Fusarium graminearum, Fusarium solani, Fusarium oxysporum, Fusarium moniliforme, Fusarium nivalum, Fusarium umbilicale.

[0055] Such as figure 2 as shown, figure 2 The label "1" in refers to the detection result of Gibberella Fujikura (positive control), figure 2 The mark "2" in refers to the test result of Fusarium graminearum, figure 2 The mark "3" in refers to the detection result of Fusarium graminearum, figure 2 The label "4" in refers to the test result of Fusarium solani rot, figure 2 The label "5" in refers to the test result of Fusarium oxysporum, figure 2 The label "6" in refers to the test r...

Embodiment 3

[0058] Sensitivity Detection of RPA Primers of Gibberella Fujikura, the Pathogen of Rice Bakanae Disease

[0059] With reference to the method for obtaining the RPA amplification product of rice bakanae disease pathogen Gibberella Fujikura, wherein primers select forward primer Ff-RPA-F, reverse primer Ff-RPA-R, wherein the genomic DNA of the former Gibberella Fujikura, according to 10 Doubling serial dilutions were 10ng / μL, 1pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL.

[0060] Such as image 3 As shown, the labels "100fg", "1pg", "10pg", "100pg", "1ng", and "10ng" respectively refer to different concentrations (100fg μL, 1pg / μL, 10pg / μL, 100pg / μL, 1ng / μL, 10ng / μL) Genomic DNA detection results.

[0061] The above detection result graph shows that the lower detection limit of the DNA of Gibberella fujikura, the pathogen of rice bakanae disease, is 10 pg DNA / μL; this shows that the RPA primers and probes of the present application have higher detection sensitivity.

[0062] I...

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Abstract

The invention discloses an RPA primer for detecting gibberella fujikuroi of a rice bakanae disease and a detection method and belongs to the technical field of biosafety. The RPA primer is as follows:an upstream primer sequence Ff-PRA-F of the RPA is as shown in SEQ ID No.1, and a downstream primer sequence Ff-PRA-R of the RPA is as shown in SEQ ID No.2. The invention further provides a detectionmethod formed on the basis of the RPA primer. An RPA technology is applied to molecular detection of the gibberella fujikuroi for the first time; the RPA primer has the characteristics of being highin specificity, high in sensitivity, good in practicability and relatively low in requirements for instruments and equipment; and a novel method is provided for early diagnosis of the rice bakanae disease, disease control and pesticide use in a reduced amount.

Description

technical field [0001] The invention belongs to the technical field of biological safety, and in particular relates to an RPA primer and a detection method for detecting gibberella fujikura, the pathogen of rice bakanae disease. Background technique [0002] The rice bakanae disease caused by Fusarium fujikuroi is a systemic infection disease of rice, and the field with severe disease can reduce the yield by more than 50%. In addition, Gibberella fujikura will produce a large amount of toxins during the infection process, causing food safety problems. The traditional detection methods of Gibberella Fujikura mainly include morphological identification and molecular detection based on PCR technology, but because these technologies require professional morphological knowledge, or long diagnostic reagents, or sophisticated scientific instruments, they are not suitable for non-professionals Therefore, it is of great significance to establish a rapid diagnosis technology for Gibb...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/04C12Q1/6844C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2522/101
Inventor 迟元凯赵伟戚仁德叶梦迪崔晓芳汪涛
Owner PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
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