Method for detecting banana bunchy top virus by utilizing IC-LAMP

A technology of banana bunchy top virus and IC-LAMP, which is applied in the field of plant pathogen detection, can solve problems such as poor sensitivity, and achieve the effects of high sensitivity, improved detection efficiency, and simplified operation process

Inactive Publication Date: 2017-04-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

ELISA is not as sensitive as PCR for det...

Method used

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  • Method for detecting banana bunchy top virus by utilizing IC-LAMP
  • Method for detecting banana bunchy top virus by utilizing IC-LAMP
  • Method for detecting banana bunchy top virus by utilizing IC-LAMP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 IC-LAMP detection of banana samples suspected of BBTV infection collected in the field

[0052] Banana samples suspected to be infected with BBTV collected in the field are used as samples to be tested, and the method of the present invention is used for IC-LAMP detection. Healthy banana plants are used as negative controls, and banana plants infected with BBTV are used as positive controls.

[0053] 1. Primer Design

[0054] Design primers according to the conserved sequence of the BBTV replicase gene obtained by NCBI, find out the conserved region sequence of the viral replicase gene after analysis by DNAstar software as a template reference sequence, and design the LAMP outer primer pair BBTV-F3 / BBTV-B3 and inner primer pair BBTV-FIP / BBTV-BIP, the sequence is as follows:

[0055] BBTV-F3: 5'-GGCGCGATATGTGGTATGC-3';

[0056] BBTV-B3: 5'-TCATACAGTACGCCCGTGC-3';

[0057] BBTV-FIP:

[0058] 5'-GTACCTCTCCTGTCCCCTCTCCACCAACAATCCCGCTTCACTTCC-3';

[0059] ...

Embodiment 2

[0073] Example 2 Sensitivity Comparison of PCR and IC-LAMP Detection of BBTV

[0074] 1. In order to compare the sensitivity of detecting BBTV by PCR and IC-LAMP methods, an appropriate amount of BBTV-infected bananas was mixed and divided into two parts. One part was extracted with the plant tissue DNA extraction kit of Omege Company to extract the total DNA, the total DNA concentration was adjusted to 100ng / μL, and the initial nucleic acid concentration was determined to be 8.40×10 by quantitative analysis. 6 copies / μL, perform 10-fold concentration serial dilution (8.40×10 6 ~8.40×10 -1 copies / μL) for PCR amplification; another sample of the same concentration was used for IC-LAMP amplification. Experiments were repeated 3 times.

[0075] 2. Sensitivity results of IC-LAMP detection of BBTV-infected banana samples

[0076] Capture with BBTV antiserum, and react with the IC-LAMP detection system in Example 1.

[0077] The results showed that under UV light, the d...

Embodiment 3

[0089] Example 3 IC-LAMP detects specificity of BBTV

[0090] 1. In order to analyze the specificity of detecting BBTV by PCR and IC-LAMP methods, bananas infected with CMV, BBTV or BSV virus, and the pathogenic bacteria physiological race 1 of banana fusarium wilt ( Fusarium oxysporum f. sp. Cubense race 1, Foc1), Fusarium wilt Physiological race 4 ( Fusarium oxysporum f. sp. Cubense race 4, Foc4), the pathogen of banana bacterial soft rot ( Dickeya zeae ) strain XJ8-3 and the pathogen of banana leaf sheath rot ( Dickeya dadantii ) bananas of strain XJ5-1 were used as samples, which were subjected to IC-LAMP amplification and PCR verification respectively. Experiments were repeated 3 times.

[0091] 2. The specificity test results of IC-LAMP detection of BBTV

[0092] In order to analyze the specificity of IC-LAMP for detecting BBTV, bananas infected with CMV, BBTV or BSV viruses, and bananas infected with Fusarium wilt race 1 (Foc1), Fusarium wilt race 4 (Foc4)...

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Abstract

The invention discloses a method for detecting the banana bunchy top virus by utilizing IC-LAMP and also discloses an LAMP primer set. The primer set comprises an inner primer pair and an outer primer pair. The outer primer pair is BBTV-F3/ BBTV-B3 and the sequence of the outer primer pair is show in SEQ ID NO.1-2. The inner primer pair is BBTV-FIP/ BBTV-BIP and the sequence of the outer primer pair is show in SEQ ID NO.3-4. According to the technical scheme of the invention, the serology and the LAMP are combined. Firstly, a virus particle is captured by a specific BBTV antiserum, and then the LAMP amplification and the BBTV detection are conducted with the virus particle as a template by using the above primer set. The method provides a simple, sensitive, specific and rapid detection technology for detecting BBTV in banana plants, absorptive buds and bud tissue culture seedlings. The method is simple in operation, good in specificity and high in sensitivity. The method can be applied to the detection and the identification of BBTV to monitor the occurrence, the diffusion and the propagation of BBTV.

Description

technical field [0001] The invention belongs to the technical field of detection of plant pathogens, and in particular relates to a method for detecting banana bunchy top virus ( Banana bunchy top virus , BBTV) method. The method is suitable for rapid detection and disease diagnosis of banana bunchy top virus on banana tissue culture seedlings and field banana plants. Background technique [0002] Banana bunchy top virus ( Banana bunchy top virus , BBTV) belongs to the genus Banana Bunchy Top Virus in the Dwarf Viridae family and is the pathogen of banana bunchy top disease. BBTV can only be controlled by the banana cross aphid ( Pentalonia nigronervosa ) is transmitted in a persistent manner, and its short-distance transmission mainly depends on aphids, while the long-distance transmission mainly comes from diseased propagation materials; its host range is relatively narrow, and they all belong to the Musaceae family. The diseased plants of banana bunchy top disease ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6804C12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2565/125C12Q2527/125
Inventor 饶雪琴李华平罗宝花刘娟
Owner SOUTH CHINA AGRI UNIV
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