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Kit for rapidly detecting peach bacterial shot-hole disease in fields and application thereof

A technology for bacterial and perforating diseases, applied in the field of rapid detection of plant pathogens, can solve the problem that it is difficult to analyze multiple components at the same time

Inactive Publication Date: 2020-03-31
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, for the diagnosis of diseases, there are mainly ATP fluorescence detection method, enzyme-linked immunosorbent assay (ELISA), biosensor technology, gene chip technology and the detection technology of PCR which are widely used at present. The selectivity of the reagent is high, but it is difficult to analyze multiple components at the same time, and there is a certain degree of cross reaction to compounds with similar structures

Method used

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  • Kit for rapidly detecting peach bacterial shot-hole disease in fields and application thereof
  • Kit for rapidly detecting peach bacterial shot-hole disease in fields and application thereof
  • Kit for rapidly detecting peach bacterial shot-hole disease in fields and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Embodiment 1. A LAMP loop-mediated isothermal amplification kit for peach bacterial perforator disease;

[0069] Described kit preferably comprises reaction base liquid (20 mmol / L Tris-HCl (pH=8.8), 20 mmol / L KCl, 20 mmol / L (NH 4 ) 2 SO 4 , 1% Tween-20, 3 mol / L Betaine, 2.5 mmol / L dNTPs, 15 mmol / L MgSO 4 ), Bst DNA polymerase, outer primers F3 and B3, inner primers FIP and BIP, SYBR Green I. The specific sequences of each primer are as follows:

[0070] Forward outer primer F3: 5'-CACTGCGGATTGTTACACGT-3';

[0071] Reverse outer primer B3: 5'-TGATGCCCCTCAAGAGAGG-3';

[0072] Forward inner primer FIP: 5'-TCGGTGGGTCGAATAGGTACCAGGGTGTGGAGTTGGTCGT-3';

[0073] Reverse inner primer BIP: 5'-TACGGGATCGAGACACCTTGGTCGGTGCATGGTAGATCACAT-3'.

[0074] Wherein, the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, and the reverse outer primer B3 constitute a LAMP detection primer composition for detecting peach bacterial perforation disease. ...

Embodiment 2

[0075] Example 2. Specificity test of peach bacterial perforator LAMP reaction.

[0076]In order to verify the specificity of the LAMP method, 4 kinds of pathogenic bacteria were used as the test materials. The LAMP detection results showed that the peach bacterial perforator strains could all have a yellow-green positive reaction or a ladder-like band of LAMP in agarose gel electrophoresis. The remaining 3 pathogenic bacteria showed orange negative reactions or agarose gel electrophoresis without amplified bands. Select the DNA of bacteria different from peach bacterial perforator (XCC, PST, W10, FD6) as a template, take 1 μL of the DNA solution, and carry out the LAMP reaction according to the system in Example 1. The reaction procedure is: water bath at 65°C, incubate for 45 min. The results show that based on the color reaction of the reaction system as the result judgment standard, the DNA template of peach bacterial perforation disease is yellow-green; the amplified DNA ...

Embodiment 3

[0077] Example 3. Sensitivity test of peach bacterial perforator LAMP reaction.

[0078] In order to determine the sensitivity of the LAMP detection method, the extracted DNA of peach bacterial perforator was diluted 10 times with DEPC water and stored at -80°C for future use. The DNA dilutions of various concentrations after gradient dilution were used as templates, and the system of Example 1 was subjected to LAMP reaction. The reaction procedure was: incubate in a water bath at 65°C for 45 min. Take 3 μL of the amplified product, and perform electrophoresis detection on a 1% agarose gel. The results show that the LAMP method can detect a concentration of 14.2×10 -5 μg DNA; SYBR Green I color reaction showed that the sensitivity of LAMP reaction also reached 14.2×10 -5 μg ( figure 2 ). The DNA dilutions of each concentration after gradient dilution were used as templates for PCR, and the results showed that the detected concentration of PCR was 14.2×10 -3 μg( image 3 ...

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Abstract

The invention, which relates to the technical field of biology, particularly discloses a kit for rapidly detecting the peach bacterial shot-hole disease in fields and application thereof. The kit comprises a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a reaction base solution. The detection method comprises the following steps: detecting total nucleic acid of a to-be-detectedsample; carrying out LAMP reaction by utilizing the kit without extracting plant tissue DNA; and judging whether the to-be-detected sample is infected by pathogenic bacteria or not by utilizing fluorescent dye. According to the kit, the peach bacterial perforation disease is detected by using LAMP (loop-mediated isothermal amplification). The kit has advantages of being low in cost, easy to operate, simple, rapid, high in sensitivity, high in specificity and the like. Whether the peach bacterial perforation disease is infected or not can be determined before the disease occurs to decide whether to take measures to make prevention in advance, so that a good prevention effect is realized.

Description

technical field [0001] The invention relates to a kit for rapid detection of peach bacterial perforation disease in the field and an application thereof, belonging to the technical field of rapid detection of plant pathogenic bacteria. Background technique [0002] The pathogen of peach bacterial perforation disease is the pathogenic variety of Prunus xanthomonas ( Xanthomonas arboricola pv. Pruni , Xap ). It is widely spread in many countries in Europe, and also commonly occurs in peach producing areas in my country. It mainly harms peach leaves, branches, and fruits. It is one of the important bacterial diseases on peach trees. The EU and EPPO regions list it as a Quarantine pathogens. The emergence and prevalence of diseases are greatly affected by the prevailing climatic conditions and host cultivars. Japanese nectarines and peaches are very sensitive to the disease, and bacterial perforator disease has occurred and harmed in all major production areas in my countr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12R1/64
CPCC12Q1/689C12Q1/6844C12Q2531/119
Inventor 朱峰朱鹏翔纪兆林
Owner YANGZHOU UNIV
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