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Molecular identification primer and identification method of angular leaf spot bacteria of tobacco

A technology of angular spot bacteria and identification method, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve the problem of poor specificity of biological detection methods of tobacco angular spot bacteria and serological identification methods. High cost, easy to cause false positives and other problems, to meet the needs of scientific research and production, good practicability, and strong primer specificity

Active Publication Date: 2014-12-17
中国烟草总公司黑龙江省公司烟草科学研究所
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Problems solved by technology

[0005] The purpose of the present invention is to provide specific molecular identification of Xanthomonas tabacum in view of the problems of poor specificity, low sensitivity, long cycle, high cost of serological identification methods and easy false positives in the biological detection methods of Xanthomonas tabacum in the prior art Primers and a reliable, specific and sensitive method for rapid molecular identification of Xanthomonas tabacum

Method used

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  • Molecular identification primer and identification method of angular leaf spot bacteria of tobacco
  • Molecular identification primer and identification method of angular leaf spot bacteria of tobacco
  • Molecular identification primer and identification method of angular leaf spot bacteria of tobacco

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Identification of Bacterial Isolates from Suspected Samples of Tobacco Angular Spot

[0033] (1) Isolation and culture of pathogenic bacteria from suspected samples of tobacco angular leaf spot

[0034] Collect suspected tobacco angular spot disease leaves from tobacco fields (see figure 1 ), use sterilized scissors to cut off the suspected diseased leaf spot (1-2) cm of tobacco angular leaf spot. In the ultra-clean workbench, disinfect the surface of the lesion with 70% ethanol for 30s-60s, rinse (3-4) times with sterile water, place it in LB solid medium for 2-3 days at 25°C, and collect the growths around the lesion. Put the bacterial colonies out in sterile 1000mL LB liquid medium, mix well, draw 100μL of bacterial solution to coat LB solid plate, culture at 25°C for 2-3 days, pick a single colony in 100μL LB liquid medium , and cultivated at 25°C for 4h for PCR detection.

[0035] (2) Design of primers for PCR identification of Xanthomonas tabacum

[...

Embodiment 2

[0044] Example 2 Comparison of Molecular Identification Result and Inoculation Identification Result of Tobacco Angular Spot Pathogen

[0045] Choose 3 to be identified as positive bacterium and 3 negative bacteriums through PCR, NC89 flourishes for a long time, inoculate each bacterial bacterium liquid (OD600 value is at 0.3-0.5, dilute 10 times with clear water during inoculation) with high-pressure watering can, every process inoculates 3, Investigate and record the pathogenicity of each strain about 20 days after inoculation. The statistics of the results are shown in Table 2. Test pictures see image 3 .

[0046] Table 2. Validation of PCR identification method for tobacco horn spot pathogen

[0047]

[0048]

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Abstract

The invention discloses a molecular identification primer and an identification method of angular leaf spot bacteria of tobacco. The molecular identification primer of the angular leaf spot bacteria of tobacco is as shown in SEQ ID NO: 1-SEQ ID NO: 2, wherein the sequence as shown in the SEQ ID NO: 1 is an upstream primer for identifying the angular leaf spot bacteria of tobacco and the sequence as shown in the SEQ ID NO: 2 is a downstream primer for identifying the angular leaf spot bacteria of tobacco. The molecular identification primer disclosed by the invention can be applied in specific identification of the angular leaf spot bacteria of tobacco. In addition, the invention also provides a reliable, strong-specificity and high-sensitivity rapid molecular identification method of the angular leaf spot bacteria of tobacco.

Description

technical field [0001] The invention relates to a primer and an identification method for molecular identification of Xanthomonas tabacum, belonging to the field of detection of plant pathogens. Background technique [0002] Tobacco is one of the important economic crops in our country. Tobacco angular leaf spot (Pseudomonas syringae pv. angulata) is a major bacterial disease of tobacco, which occurs seriously in northern my country and causes a certain degree of harm and economic loss every year. [0003] The classification and identification of traditional bacteria is mainly based on morphological characteristics, pathogenicity determination, physiological and biochemical characteristics, etc. The work is cumbersome and takes a long time. The identification is also susceptible to interference from many factors such as human and environmental factors, and it cannot be directly detected from plant tissues. It is difficult to meet the rapid, sensitive and stable detection re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/689
Inventor 万秀清乔婵李若李丽杰王春军元野贺国强颜培强李尊强郭思达孙宏伟焦玉生李恒全邱恩建刘德育陈荣平黄磊张海霞刘伟魏继承
Owner 中国烟草总公司黑龙江省公司烟草科学研究所
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