Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof

A citrus canker bacterium, detection column technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the lack of obvious symptoms of disease-resistant or non-susceptible varieties, complicated biochemical identification of citrus canker sores, and other diseases Confusion and other problems, to achieve high accuracy, high sensitivity, and good primer specificity

Inactive Publication Date: 2010-05-26
中华人民共和国湖南出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the quarantine of citrus canker is mainly based on the observation of the symptoms of the plants, which can be judged by the naked eye under normal circumstances, but many disease-resistant or non-susceptible varieties lack obvious symptoms and are easy to be confused with other diseases. There are many types and variants, and the biochemical identification of X. citrus canker is very complicated and difficult.
At present, there is no research report on rapid and accurate detection of citrus canker using double PCR combined with high performance denaturing liquid chromatography (doublx PCR-DHPLC)

Method used

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  • Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof
  • Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof
  • Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Primer design and synthesis

[0027] According to the reported region sequence of Xanthomonas axonopodis pv.citri OPM-12 SCARfragment (gb|AF312370.1) of Xanthomonas axonopodis pv.citri, the primer sequences were designed as follows:

[0028] SEQ ID NO.1 (forward): 5'-ACGCTCGATCTGCACCTATT-3';

[0029] SEQ ID NO. 2 (reverse): 5'-CCAACGAGTCTCAAAGCATCA-3'.

[0030] According to the reported primers, the primer sequences are:

[0031] SEQ ID NO.3 (forward): 5'-CGCCATCCCCACCACCACCACGAC-3';

[0032] SEQ ID NO. 4 (reverse): 5'-AACCGCTCAATGCCATCCACTTCA-3'.

Embodiment 2

[0034] Collection of bacteria

[0035] Colonies grown on the plate: Pick at least 5 colonies directly into a 1.5mL centrifuge tube with the inoculation loop, add 1mL sterilized water to shake fully, centrifuge at 8000g for 3min, discard the supernatant, then add 1mL sterilized water to the tube, repeat three times , Take the precipitate to extract DNA, and perform double PCR-DHPLC detection.

[0036] Citrus leaves or materials: Use a 6mm hole puncher to punch 50 small holes on citrus leaves. If there are lesion materials, take the lesion parts, or take about 1g of diseased tissue materials and add 1mL sterilized distilled water to fully shake and wash. The liquid was centrifuged at 8000 g for 5 min, the supernatant was discarded, and the precipitate was taken to extract DNA, which was detected by double PCR-DHPLC.

Embodiment 3

[0038] Bacterial DNA Extraction

[0039] 1) Add 600 μL of TE buffer, 30 μL of 10% SDS solution, and 15 μL of 20 mg / mL proteinase K to the precipitate, mix well, and incubate in a water bath at 37° C. for 1 h.

[0040] 2) Add an equal volume of chloroform-isoamyl alcohol (24:1), and mix well. Centrifuge at 10,000 g for 5 min, and transfer the supernatant to a new centrifuge tube.

[0041] 3) Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix well, centrifuge at 10,000 g for 5 min, and transfer the supernatant to a new centrifuge tube.

[0042] 4) Add 0.6 times the volume of isopropanol, mix gently, precipitate at -20°C for 1 hour, centrifuge at 10,000 g for 5 minutes, and discard the supernatant.

[0043] 5) Add 1 mL of 70% ethanol to wash the precipitate, centrifuge at 8000 g, discard the supernatant, and dry in the air.

[0044] 6) Add 50 μLTE buffer to dissolve the DNA precipitate, and store it for a long time at -20°C for future use.

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Abstract

The invention relates to the biological detection technology, in particular to primers for detecting citrus canker pathogenic bacteria and a detection method thereof. The invention aims to provide the primers and method for dual PCR-DHPLC detection of the citrus canker pathogenic bacteria. The primers for detecting the citrus canker pathogenic bacteria of the invention have good primer specificity and high detection sensitivity; and the detection method of the invention has high accuracy and high sensitivity and can quickly and simply judge whether a sample has the citrus canker pathogenic bacteria or not, thereby ensuring the safety in import and export.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a primer and a detection method for detection of canker sore citrus. Background technique [0002] Citrus canker is an important quarantine disease caused by the infection of Xanthomonas axonopodis pv.Citri. It is an object of quarantine at home and abroad, seriously affecting the safe production and foreign trade of citrus, and has been listed by the General Administration of Quality Supervision, Inspection and Quarantine of China. Into the "List of Imported Plant Quarantine Pests of the People's Republic of China" published in 2007. [0003] The fungus survives the winter in the lesions of leaves, branches and fruits, and overflows from the diseased parts when the conditions are suitable in the next spring. It is spread by wind and rain and insects, and invades through the stomata, lenticels and wounds of the host. Make leaves, shoots and fruits diseased, and there is no effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/64
Inventor 朱金国陈小帆王象贤莫瑾左静
Owner 中华人民共和国湖南出入境检验检疫局
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