Specific amplification primer for detecting marssonina coronaria and detection method

A technology for apple brown spot bacteria and amplification primers, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of long detection process, high detection cost, and low detection efficiency , to save costs, improve detection efficiency, and shorten the experimental process

Inactive Publication Date: 2010-11-24
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Another object of the present invention is to provide a method for rapid molecular detection of the apple brown spot pathogen by using the specific amplification primer, which solves the problems of long detection process, low detection efficiency and high detection cost in the prior art

Method used

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  • Specific amplification primer for detecting marssonina coronaria and detection method
  • Specific amplification primer for detecting marssonina coronaria and detection method
  • Specific amplification primer for detecting marssonina coronaria and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] In July 2007, samples collected from apple brown spot disease in Baishui County, Shaanxi Province were amplified. The specific operation steps are as follows:

[0037] step 1

[0038] Select the diseased leaves of the orchard, wash and dry them, use a sterilized insect needle to pick out conidia discs under a stereo microscope and transfer them to sterilized white paper sheets for later use;

[0039] Step 2, prepare 4 PCR reaction systems according to the following system:

[0040] 10×Taq Buffer 6μL

[0041] DNTP 0.2mM

[0042] P 1 1μM

[0043] P 2 1μM

[0044] Taq DNA Polymerase 1U

[0045] Mg 2+ concentration 1.75mM

[0046] DTT concentration 1.25mM

[0047] Add sterilized ultrapure water to make up the reaction system to 50 μL;

[0048] Among them, P 1 and P 2 According to the DNA sequence of the pathogenic bacteria of apple brown spot, a pair of specific amplification primers were designed for th...

Embodiment 2

[0072] In August 2008, the bacteria of brown spot of apples, roses and poplars collected in Yangling, Shaanxi were amplified. The specific steps are as follows:

[0073] step 1

[0074] After the collected infected leaves of apple, Chinese rose and poplar are washed and dried, use a sterilized insect needle to pick out conidia disks under a stereo microscope and transfer them to sterilized white paper sheets for later use;

[0075] Step 2, prepare 13 PCR reaction systems:

[0076] 10×Taq Buffer 6μL

[0077] DNTP 0.2mM

[0078] P 1 1μM

[0079] P 2 1μM

[0080] Taq DNA Polymerase 1U

[0081] Mg 2+ concentration 1.75mM

[0082] DTT concentration 1.25mM

[0083] Add sterilized ultrapure water to make up the reaction system to 50 μL;

[0084] Among them, P 1 and P 2 It is a pair of specific amplification primers designed according to the DNA sequence of the pathogenic bacteria of apple brown spot, the upstream ...

Embodiment 3

[0106] Select apple brown spot, ring pattern, anthracnose, leaf spot, these common apple disease pathogens for amplification and comparison. The specific steps are as follows:

[0107] step 1,

[0108] The samples of common apple diseases such as apple leaf spot disease, apple ring spot disease, apple anthracnose disease, and apple leaf spot disease were selected, and the bacterial cells of the diseased parts were picked under a stereomicroscope with a sterilized insect needle and transferred to Sterilized white paper sheets are available for use.

[0109] Step 2, prepare 13 PCR reaction systems:

[0110] 10×Taq Buffer 6μL

[0111] DNTP 0.2mM

[0112] P 1 1μM

[0113] P 2 1μM

[0114] Taq DNA Polymerase 1U

[0115] Mg 2+ concentration 1.75mM

[0116] DTT concentration 1.25mM

[0117] Add sterilized ultrapure water to make up the reaction system to 50 μL;

[0118] Among them, P 1 and P 2 It is a pair of sp...

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Abstract

The invention discloses a specific amplification primer for detecting marssonina coronaria. A method for quickly obtaining gene segments of the marssonina coronaria by using the specific primer aims to prevent obstruction brought to actual researches by the difficult separation of pathogenic bacteria. The invention also discloses a rapid molecular detection method for the marssonina coronaria by using the specific amplification primer, which comprises the following steps of: first collecting field diseased leaves and picking and placing the acervulus of the marssonina coronaria on a white paper sheet with a sterilized insect needle; then preparing a polymerase chain reaction (PCR) system; next transferring the acervulus into a small PCR amplification tube in which the PCR system is arranged and performing centrifugation to completely immerse the acervulus in the prepared PCR system; and finally performing PCR amplification and detecting the amplification product. By changing the PCR amplification reaction system and the amplification procedure, the target segment of the pathogenic bacteria can be directly amplified. The primer and the method prevent the separation of the pathogenic bacteria and additional DNA extraction, shorten a test flow and improve the detection efficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, relates to a specific amplification primer for detecting apple brown spot fungus, and also relates to a rapid molecular detection method for apple brown spot fungus by using the specific amplification primer. Background technique [0002] Apple Brown Spot Marssonina coronaria (Ellis & J.J. Davis) J. J. Davis, synonym M. mali (Henn.) S. Ito (Sexual Generation Diplocarpon mali Harada & Sawamura) is one of the major diseases that cause early defoliation of apple trees. Apple brown spot disease not only harms the leaves and petioles, but also causes the disease of apple fruits during storage, which seriously affects the yield and quality of apples. In recent years, this disease has a tendency to increase year by year, and more and more people pay attention to it. [0003] Accurately grasping the biological characteristics of pathogens and revealing the genetic characteristics of path...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 孙广宇朱刚张荣张利娜
Owner NORTHWEST A & F UNIV
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