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Real-time fluorescent quantitation PCR (Polymerase Chain Reaction) method for detecting Chinese cabbage verticillium pathogenic bacteria

A real-time fluorescence quantitative and Chinese cabbage technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of not being able to use large batches of detection practices and effectively distinguish Verticillium dahliae related species of bacteria

Active Publication Date: 2014-06-25
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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AI Technical Summary

Problems solved by technology

Traditional pathogen content detection usually uses colony counting method, such as NP-10 plate detection method, but this method takes about two weeks to isolate pathogenic bacteria from diseased strains, so it cannot be used in large-scale detection practice; secondly, this method cannot be effective Distinguishing Relative Species of Verticillium dahliae

Method used

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  • Real-time fluorescent quantitation PCR (Polymerase Chain Reaction) method for detecting Chinese cabbage verticillium pathogenic bacteria
  • Real-time fluorescent quantitation PCR (Polymerase Chain Reaction) method for detecting Chinese cabbage verticillium pathogenic bacteria
  • Real-time fluorescent quantitation PCR (Polymerase Chain Reaction) method for detecting Chinese cabbage verticillium pathogenic bacteria

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Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1, primer design

[0051] 1. Primer design

[0052] According to the specific rDNA-ITS sequence (NCBI accession number: JN564038) of Verticillium dahliae pathogenic bacteria, the specific amplification primers are designed as follows:

[0053] HW1-F: 5'-GTTACAGCTCGCATCGGAGT-3' (SEQ ID No. 1)

[0054] HW1-R: 5'-CAGCGGGTATTCCCTACCTGA-3' (SEQ ID No. 2)

[0055] The length of the amplified target fragment is 100bp.

[0056] 2. Primer specificity and sensitivity detection

[0057] (1) Primer specificity detection

[0058] Extract the leaf tissue of Chinese cabbage not infected with the pathogen of Verticillium wilt of Chinese cabbage, the leaf tissue of Chinese cabbage infected with downy mildew of Chinese cabbage, the pathogen of Chinese cabbage Verticillium wilt BCHW10-2, the disease of Chinese cabbage infected with the pathogen of Verticillium wilt of Chinese cabbage DNA from leaves, diseased stems and roots, and expressed as ddH 2 O is a blank control. ...

Embodiment 2

[0069] Embodiment 2, fluorescent quantitative PCR method detects Verticillium dahliae pathogenic bacteria in the sample

[0070] 1. Sample preparation

[0071] Greenhouse sample: The seedlings of various varieties of Chinese cabbage that have grown to three leaves and one heart are pulled out from the nutrient pot (natural root injury), and the spore suspension of Chinese cabbage verticillium wilt pathogen BCHW10-2 (concentration is 1 × 10 7 seedlings / mL) soaked the roots of seedlings for 15 minutes, and then replanted the seedlings in a 6×6cm nutrient pot, and inoculated sterile water as a control; The plants inoculated with pathogenic bacteria were sampled, and the plants inoculated with sterile water were used as the control group. Five plants were taken from each group of materials, and the roots, stems and leaves were stored separately, and freeze-dried in an ultra-low temperature vacuum freezer for later use.

[0072] Field samples: the leaves of each variety of cabbag...

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Abstract

The invention discloses a real-time fluorescent quantitation PCR (Polymerase Chain Reaction) method for detecting Chinese cabbage verticillium pathogenic bacteria. A pair of primers disclosed by the invention consists of a DNA (Desoxvribose Nucleic Acid) molecule shown as SEQ ID No.1 and a DNA molecule shown as SEQ ID No.2. The method disclosed by the invention is a rapid, accurate and efficient verticillium-resistant molecular diagnosis system, and a good foundation is laid for the screening and resistant transformation of Chinese cabbage verticillium resources. Moreover, the method can be used for predicting and forecasting Chinese cabbage verticillium, and has important significance to the safe production of Chinese cabbage.

Description

technical field [0001] The invention relates to a real-time fluorescent quantitative PCR method for detecting the pathogenic bacteria of verticillium wilt of Chinese cabbage. Background technique [0002] Verticillium wilt is mostly a soil-borne vascular disease caused by root infection of Verticillium dahliae and Verticillium black and white of Verticillium genus Verticillium. The host range of the disease is wide and there are different physiological races. Microsclerotia survives in the soil for a long time and has a high ability to resist adverse environments. It is difficult to prevent and control. It has seriously affected cotton, tomato, pepper, eggplant and other crops, and is called the "cancer" of plants. [0003] Chinese cabbage belongs to the Brassicaceae Brassica genus. It is the vegetable crop with the largest cultivated area in my country and has gradually become a worldwide vegetable crop. According to statistics from the Ministry of Agriculture, the annual ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/04C12Q1/6851C12Q1/686C12Q2561/113C12Q2545/114
Inventor 于拴仓苏同兵陈娟张凤兰汪维红余阳俊张德双赵岫云
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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