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QLAMP (quantitative loop mediated isothermal amplification) method for quantitatively monitoring infection and propagation of Chinese walnut dry rot and used primers

A pecan dry rot and quantitative loop technology, applied in the fields of plant disease epidemiology, dynamic monitoring and early warning, biotechnology and plant disease epidemiology, can solve the problems of complicated detection process, low detection sensitivity, and long detection time, etc. Achieve the effect of stable reaction, high sensitivity and optimized reaction temperature

Active Publication Date: 2019-06-18
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] With the continuous development of nucleic acid-related identification methods, methods based on common PCR have been successfully used to detect hickory dry rot pathogens. The process is complicated, and the key is that it is also susceptible to various PCR inhibitors

Method used

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  • QLAMP (quantitative loop mediated isothermal amplification) method for quantitatively monitoring infection and propagation of Chinese walnut dry rot and used primers
  • QLAMP (quantitative loop mediated isothermal amplification) method for quantitatively monitoring infection and propagation of Chinese walnut dry rot and used primers
  • QLAMP (quantitative loop mediated isothermal amplification) method for quantitatively monitoring infection and propagation of Chinese walnut dry rot and used primers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the specificity analysis of detection system

[0078] Botryosphaeria dothidea was used as a positive control, Trichoderma viride, Fusarium proliferatum, Pestalotiopsis microspora, Nigrospora chinensis, chain Alternaria regonensis and Daldinia sp. were used as negative controls, and double distilled water was used as blank control for testing.

[0079] A total of 19 μL of the detection solution was added, and 1 μL of the DNA template to be tested (concentration between 0.001 ng / μL and 100 ng / μL, both positive and negative controls, 0.1 ng / μL) was added to form a 20 μL detection reaction system. The 20 μL system contains 8 U / μL BstDNA polymerase 1 μL, 10×ThermoPol Buffer 2.0 μL, 20 μM FIP primer 1.6 μL, 20 μM BIP primer 1.6 μL, 10 μM F3 primer 0.25 μL, 10 μM B3 primer 0.25 μL, 25 mM MgCl 25.0 μL, 2.0 μL of 10 mM dNTPs, 2.4 μL of 5M betaine, 1.2 μL of 10×SYBR Green I, 1 μL of DNA template, and make up to 20 μL with double distilled water. The q-LAMP amplif...

Embodiment 2

[0081] Embodiment 2, the sensitivity detection of primer

[0082] With the dry rot pathogen q-LAMP detection system after optimization (as described in embodiment 1) to the DNA solution detection result of the dry rot pathogen pure hyphae of gradient dilution as follows Figure 4 As shown, when the DNA template concentration is 100ng / μL, 10ng / μL, 1ng / μL, 0.1ng / μL, 0.01ng / μL and 0.001ng / μL, it can be effectively amplified, and the repeatability of the same sample is reliable. The amplification time corresponding to each concentration is 16.32min, 18.68min, 22.43min, 26.63min, 30.84min and 33.26min respectively. The results showed that the theoretical detection line of dry rot pathogen DNA was 1pg DNA in the established q-LAMP detection system.

Embodiment 3

[0083] Embodiment 3, the acquisition of standard curve:

[0084] Standard curve 1, when the spore concentration is 1.0×10 5 pcs / mL, 1.0×10 4 pcs / mL, 1.0×10 3 pcs / mL, 1.0×10 2 / mL and 1.0×10 1 When samples / mL, the amplification time corresponding to each concentration is 16.67min, 21.51min, 28.69min, 36.26min and 43.61min, according to the dilution sample C T Standard curve between the value (y) and the Log value (x) of the number of spores per milliliter of sample: y=-6.863x+49.937, correlation coefficient R 2 =0.9922, the calculation formula for the number of spores (pieces) is

[0085] Calculation formula: construct a standard curve, use the Log value of the number of spores per milliliter as the x-axis, and the corresponding C T The value is plotted on the y axis, showing a decreasing linear equation [y=mx+b, or y=m (log number of spores per milliliter of sample)+b], the following equation can be deduced from the decreasing linear equation to determine Unknown samp...

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Abstract

The invention discloses qLAMP (quantitative loop mediated isothermal amplification) primers used for monitoring quantity dynamics of pathogen spores of Chinese walnut dry rot. The q-LAMP primer composition comprises following four primers: an upstream outer primer (F3), a downstream outer primer (B3), an upstream inner primer (FIP) and a downstream inner primer (FIB). The invention further provides a q-LAMP kit containing the q-LAMP primer composition and provides a q-LAMP method for performing the quantity dynamics of the pathogen spores of Chinese walnut dry rot by the kit, and the number ofspores and DNA concentration are obtained. With the adoption of the method, the number of the pathogen spores of Chinese walnut dry rot in rainwater, trees and soil can be monitored quantitatively.

Description

technical field [0001] The invention belongs to the field of biotechnology and plant disease epidemiology, and relates to quantitative loop-mediated isothermal amplification (qLAMP) technology for monitoring the number of spores of hickory dry rot disease fungus in rainwater, tree body and soil, etc. The primer set and its application method belong to the technical field of plant disease epidemiology, dynamic monitoring and early warning. Background technique [0002] Botryosphaeria dothidea is a host-dependent weakly parasitic phytopathogenic fungus with a wide range of hosts. It is a fungal pathogen that endangers the health of woody plants worldwide. Walnut dry rot. Pecan dry rot occurs widely in the main pecan producing areas such as western Zhejiang, and the affected area is gradually expanding, which is the bottleneck of the development of the pecan industry. The pathogenic bacteria overwinter in the hickory tree with mycelium, and the pathogens latent in the tree in...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851C12Q1/06C12N15/11C12R1/645
Inventor 张传清王琼伟刘亚慧徐炳潮
Owner ZHEJIANG FORESTRY UNIVERSITY
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