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Alternaria solani PCR detection specific primer and detection method thereof

A technology of tomato early blight bacteria and specific primers, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. high sensitivity and specificity

Inactive Publication Date: 2015-12-02
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to detect and identify the tomato early blight pathogen in the prior art based mainly on morphological characteristics, time-consuming, cumbersome procedures, strong experience, low accuracy, and it is difficult to monitor and control the pathogenic bacteria in time for the occurrence of the disease In order to solve the problem of spread and prevalence of tomato early blight, a PCR detection specific primer for tomato early blight and its detection method are provided. Using the PCR detection specific primer of the present invention to detect and identify tomato early blight has high accuracy and strong specificity , high sensitivity, easy operation, short detection time and reliable results

Method used

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  • Alternaria solani PCR detection specific primer and detection method thereof
  • Alternaria solani PCR detection specific primer and detection method thereof
  • Alternaria solani PCR detection specific primer and detection method thereof

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Embodiment 1

[0027] Embodiment 1: PCR detects the specificity verification of specific primers to tomato early blight bacteria

[0028] With tomato early blight and other pathogenic bacteria as the test strains, the CTAB method was used to extract the genomic DNA of the test strains. The specific method is as follows: Take a small amount of mycelium powder in a 1.5mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle). ), add 900 μL 2% CTAB (cetyltrimethylammonium bromide) extract (2%CTAB; 100mmol / LTris-HCl, pH8.0; 20mmol / LEDTA, pH8.0; 1.4mol / LNaCl) and 90μL SDS (Sodium dodecylbenzene sulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000r.min -1 Centrifuge for 15 minutes; take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (the volume ratio of each is 25:24:1), shake and mix gently, 12000r.min ...

Embodiment 2

[0030] Embodiment 2: Sensitivity detection of specific primers to tomato early blight genomic DNA

[0031] 1. Conventional PCR amplification

[0032] Dilute the genomic DNA of the pure culture of Phytophthora infestans with sterile ultrapure water, and prepare serial concentrations of 10-fold order of magnitude for future use. Use the primer ASF / ASR described in the present invention to carry out PCR amplification to the genomic DNA of different serial concentrations, evaluate the sensitivity of this primer to the detection of tomato early blight genomic DNA, amplification reaction system and reaction procedure are as follows: PCR reaction system 25 μ L, Includes 2 x Taq PCRMasterMix (Beijing Tiangen Biochemical Technology Co., Ltd.) 12.5 μL, 10 μmol / L ASF / ASR primers 1.0 μL each, 2.0×10 -5 ~200ng DNA template, make up to 25μL with sterile ultrapure water. The amplification reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, anneal...

Embodiment 3

[0036] Embodiment 3: detection of tomato early blight bacteria in diseased leaves

[0037] DNA Extraction of Tomato Early Blight from Diseased Leaves : DNA was extracted by NaOH rapid lysis method, the specific process is as follows: add 0.5mol / L NaOH 30μL to 1.0mg of diseased leaf tissue, grind the tissue fully into a paste, transfer it to a 1.5mL centrifuge tube, centrifuge at 12,000rpm for 6min, and take the supernatant Add 495 μL of 0.1mol / LTris-HCl (pH=8.0) to 5 μl of the solution and mix well, and take 1.0 μL as a PCR template for amplification. PCR amplification detection: The primer ASF / ASR of the present invention is used for PCR amplification. PCR reaction system 25μL, including 2× Taq PCRMasterMix (Beijing Tiangen Biochemical Technology Co., Ltd.) 12.5 μL, 10 μmol / L ASF / ASR primers 1.0 μL each, 2.0×10-5 ~200ng DNA template, made up to 25 μL with sterile ultrapure water; amplification parameters: 95°C pre-denaturation for 5 minutes, 94°C denaturation for 30 se...

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Abstract

The invention provides an alternaria solani PCR detection specific primer which comprises an upstream primer ASF: 5'-ACCACAAGGACCAACCCATA-3' and a downstream primer ASR: 5'-CCTACCTGATCCGAGGTCAA-3'. The invention further provides an alternaria solani PCR rapid detection method by using the primer, according to the method, a to-be-tested sample genome is taken as a template, the primer is used for PCR amplification, and a 409 bp amplified fragment can be detected by using gel electrophoresis after amplification is finished. The primer has high specificity, the sensitivity is high, the operation process is easy and convenient to operate, the detection requirement for alternaria solani on agricultural production can be met, and the defects that the operation is tedious, consumed time is long, the accuracy is low and the like in a conventional detection method are overcome.

Description

technical field [0001] The invention relates to specific primers for PCR detection of tomato early blight pathogen and a detection method thereof, and belongs to the technical field of detection, identification and prevention of crop diseases. Background technique [0002] tomato( Solanum lycopersicum L.) is one of the most widely planted and high-consumption vegetable crops in the world. China is a major exporter of tomato, and it is cultivated in large areas in both the north and the south. Tomato fruit is extremely nutritious and has many varieties and types. It can be eaten as a raw fruit or as a delicacy on the table as a vegetable. It can also be processed into functional canned foods such as tomato sauce and tomato juice, creating a new era for the consumer market. Good economic benefits. With the advancement of cultivation technology, in recent years, tomatoes have been mainly produced in protected areas to supply the market. On the one hand, the excellent growth e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 赵建伟何玉仙兰成忠
Owner INST OF PLANT PROTECTION FAAS
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