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Detection kit and method for Asia erwinia amylovora

A detection kit and the technology of the kit, applied in the biological field, can solve the problems of long cycle, short cycle, difficult identification, etc., and achieve the effects of high accuracy, improved efficiency and good practicability

Inactive Publication Date: 2009-08-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Technical problem The purpose of the present invention is to solve the problems such as the long required period and difficult identification of the biological detection method of Asian amyrrhea in the prior art, and provide the detection method and detection kit thereof for Asian amylovora, which can be used for the detection of Asian amylovora. Perform PCR detection with high accuracy, short cycle and good sensitivity

Method used

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  • Detection kit and method for Asia erwinia amylovora
  • Detection kit and method for Asia erwinia amylovora

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: PCR amplification of Asian amylovora specific primers HrcC-F1 / HrcC-R1.

[0037] Asian pear fire detection kit, including the following components:

[0038] The Specific Primer Sequences of Asian Amyrrhea

[0039] Upstream primer HrcC-F1: 5′-GACGTTTGCACCTGGAAACCG-3′

[0040] Downstream primer HrcC-R1: 5′-GCGGTAGGTAATCAAGGCCAC-3′

[0041] Kit reaction system:

[0042] 1mL detection solution includes: 0.05mmol Tris Cl, 0.125mmol KCl, 0.0025mmol MgCl 2 , 0.01 mmol dNTPs, 0.25 mmol of upstream and downstream primers, 0.1 mg of BSA, 100 units of Taq DNA polymerase. The solution was stored in a -20°C freezer. The storage period is 1 year.

example 1

[0043] Example 1 Detection of Asian amylovora from infected pear branches in Zhejiang:

[0044] The method that the above-mentioned Asian amyrrhea detection kit is used for detecting Asian amylovora comprises:

[0045] 1) PCR detection of Asian amylovora

[0046] Take 1 μL of bacterial suspension, add 10 μL kit solution and 14 μL sterilized ultrapure water, the total volume is 25 μL;

[0047] The PCR amplification program was denaturation at 94°C for 3 minutes; entering cycle, denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and finally extension at 72°C for 5 minutes.

[0048] Electrophoresis detection of amplified product: take 10 μ L of PCR amplified product, carry out electrophoresis on 1% (g / 100 ml) agarose gel, the voltage is 50-100 V, and detect the result under ultraviolet light after 30 minutes; if there is a molecular weight A DNA band of about 330bp proves that the detected pathogen is the Asian am...

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Abstract

The invention relates to a test kit of Asian erwinia amylovora, belonging to the category of preventing and treating diseases of crops as well as plant quarantine. The upstream primer is HrcC-F1:5'-GACGTTTGCACCTGGAAACCG-3', and the downstream primer is HrcC-R1:5'-GCGGTAGGTAATCAAGGCCAC-3'. The kit comprises 1mL of detecting solution, containing 50mmol.L Tris.Cl, 125mmol.L KCl, 2.5mmol.L MgCl, 10mMd NTPs, 0.25mmol / mL of upstream and downstream primers, 0.1mg.mLBSA, and 100 units of Taq DNA polymerase. Based on the primers, the test kit of Asian erwinia amylovora has higher specificity, sensitivity and stability, so as to provide a fast, sensitive and special technical method for testing the Asian erwinia amylovora. The products of special PCR of the Asian erwinia amylovora are shown in the figure.

Description

(1) Technical field [0001] The invention relates to a detection kit for Erwinia pyrifoliae and a detection method thereof, belonging to the field of biotechnology. It is specially used for the high-sensitivity and rapid detection of Asian amylovora pathogens carried by customs in and out of Asian pears, and can also be used for early diagnosis and monitoring of Asian amylovora in the field. (2) Background technology [0002] Since 1995, a disease very similar to pear fire blight (Erwinia amylovora) has been found on Asian pears (Pyrus pyrifoliae) in orchards in the Chuncheon area of ​​Korea. Subsequent studies have shown that the disease is very similar to, but not identical to, Amyrrha blight. The fungus mainly infects Asian pears and some Western pear (Pyruscommunis) varieties, and has no pathogenicity to apples. Molecular identification in Germany further identified the disease as a new species: Erwinia pyrifoliae. This disease does not occur in our country, and it is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/18
Inventor 胡白石田艳丽张蕾刘凤权
Owner NANJING AGRICULTURAL UNIVERSITY
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