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Primer pair and probe for detecting Phytophthora syringae and detection method thereof

A technology of Phytophthora syringae and primer pairs, which is applied in the field of molecular biology detection to achieve the effects of good specificity, high sensitivity and easy operation

Inactive Publication Date: 2016-01-20
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In recent years, real-time fluorescence quantitative PCR (fluorescence RT-PCR) technology and its related molecular detection technology have been widely used in the molecular detection and early warning of the growth and decline of plant pathogens. report

Method used

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  • Primer pair and probe for detecting Phytophthora syringae and detection method thereof
  • Primer pair and probe for detecting Phytophthora syringae and detection method thereof
  • Primer pair and probe for detecting Phytophthora syringae and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0023] The design and synthesis of embodiment 1 primer pair and probe

[0024] According to the Phytophthora syringae ITS sequence, the primer pair and probe were designed using the probe design software Express3.0. The primer pair sequence is:

[0025] PS-F (forward primer): 5'-GTCTGGCTTCGGCTGGAT-3'

[0026] PS-R (reverse primer): 5'-AGTGGGCTACTAGCTCCAGGC-3'

[0027] The sequence of the probe is: 5'-F1-TGGCGACCGCTCTGA-Q1-3'; wherein, F1 is a FAM fluorescent dye, and Q1 is a non-fluorescent quenching group MGB.

[0028] The above primer pairs and probe sequences were synthesized by Shanghai Jikang Biotechnology Co., Ltd. The extraction of embodiment 2 sample total DNA

Embodiment 2

[0029] A proper amount of Phytophthorasyringae fungal mycelia or diseased fruits and leaves were collected, and DNA was extracted with Thermo Fisher Plant Direct Extraction Kit (ThermoPhireDirect PCR Kit).

Embodiment 3

[0030] Example 3 Establishment of real-time fluorescence quantitative PCR (fluorescence RT-PCR) method

[0031] 1. Fluorescence RT-PCR reaction system

[0032] Using the total DNA as a template, carry out real-time fluorescent PCR reaction, the reaction system (total volume 25μL) is: sample DNA 1μL (0.0005-50ng), 10×PCR reaction buffer (without Mg 2+ ) 2.5 μL, 25 mM MgCl 2 2.0 μL, 2.5 mM dNTP 2.0 μL, 10 μM primer PS-F 1 μL, 10 μM primer PS-R 1 μL, 10 μM probe 1.5 μL, 5U / μL Taq DNA polymerase 0.3 μL, ddH 2 O13.7 μL.

[0033] 2. Real-time fluorescent PCR reaction conditions

[0034] Place the sample tube into the BIO-RAD IQ TM 5. After the fluorescent PCR instrument, set the following conditions for the reaction: the first cycle is 95°C for 3min; then 95°C for 10s, 60°C for 30s, 40 cycles. Collect data after each cycle. After the reaction, judge the result according to the amplification curve.

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Abstract

The invention provides a primer pair and a probe for detecting Phytophthora syringae. The primer pair has the nucleotide sequences shown as SEQ ID No.1 and 2, and the probe has a nucleotide sequence shown as a SEQ ID No.3. The invention further provides a fluorescence RT-PCR method to detect the Phytophthora syringae. The method is as below: using a sample total DNA as a template, using the primer pair and probe to conduct real-time PCR amplification, collecting data at end of each cycle, and after the reaction determining result according to an amplification curve. The method can quickly and accurately detect Phytophthora syringae from ill tissues the infected by Phytophthora syringae. A detection kit constructed according to the method has advantages of simple operation, good specificity and high sensitivity, and has important significance for early warning of Phytophthora syringae epidemic, pathogen monitoring in the epidemic area and import and export safety.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a pair of primers and a probe for detecting Phytophthora syringae and a detection method thereof. Background technique [0002] Phytophthorasyringae (Klebahn) Klebahn is an important quarantine pathogen in my country, which can cause serious and devastating diseases of ornamental plants and fruit trees. The pathogen has a wide range of hosts and mainly harms 29 genera in 14 families, such as: European clove (Syringavulgaris), citrus lemon (Citruslimon), navel orange (C. sinensis), fennel (Foeniculum vulgare), apple genus Apple (Maluspumila), sweet cherry (Prunusavium) of Prunus, pear (Pyruscommunis) of Pyrus, Pyracanthassp., etc., can cause various diseases on roots, stems, leaves and fruits. At present, there are reports of Phytophthora syringae causing harm in many countries in the Americas, Europe, Asia, and Africa. [0003] Therefore, the development of early and ra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
CPCC12Q1/686C12Q2561/113C12Q2561/101
Inventor 杜洪忠吴品珊
Owner CHINESE ACAD OF INSPECTION & QUARANTINE