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Primer pair and probe used for detecting phytophthora hibernalis and detection method for phytophthora hibernalis

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

Inactive Publication Date: 2014-04-16
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In recent years, real-time fluorescent quantitative PCR (fluorescent RT-PCR) technology and related molecular detection technologies have been widely used in the molecular detection and early warning of the growth and decline of plant pathogens. However, fluorescent RT-PCR technology is rarely used to detect Phytophthora winteri Aspects of reports

Method used

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  • Primer pair and probe used for detecting phytophthora hibernalis and detection method for phytophthora hibernalis
  • Primer pair and probe used for detecting phytophthora hibernalis and detection method for phytophthora hibernalis
  • Primer pair and probe used for detecting phytophthora hibernalis and detection method for phytophthora hibernalis

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Embodiment 1

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

[0024] According to the ITS sequence of Phytophthora winteris, the primer pair and probe were designed using the probe design software Express 3.0. The sequence of the primer pair is:

[0025] PH-F (forward primer): 5'-CGACTTGCCACCGGGA-3'

[0026] PH-R (reverse primer): 5'-AACGGTACTTCTCTTTGCTCGAA-3'

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

[0028] The above primer pairs and probe sequences were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

Embodiment 2

[0029] The extraction of embodiment 2 sample total DNA

[0030] Include the following steps:

[0031] 1) Collect an appropriate amount of Phytophthora hibernalis fungal mycelium or infected fruits and leaves, grind it into powder with liquid nitrogen, take 0.4g and put it into a 1.5mL centrifuge tube.

[0032] 2) Preheat the CTAB extract to 65°C, add 600 μL of the preheated CTAB extract to the 1.5 mL centrifuge tube containing the powder, and mix well.

[0033] 3) Immediately put in a water bath at 65°C for 30 minutes, then add 500 μL of chloroform / isoamyl alcohol (24:1, volume ratio), turn it upside down several times, and centrifuge at 13,000 g for 1 minute.

[0034] 4) Pipette about 400 μL of the supernatant into a new 2.0ml centrifuge tube, add 500 μl of chloroform / isoamyl alcohol (24:1), 500 μL of CTAB extract, mix gently, and centrifuge at 13000g for 1 minute.

[0035] 5) Take 800μl supernatant to a new 1.5mL centrifuge tube, add 500μL isopropanol, mix gently, and plac...

Embodiment 3

[0038] Example 3 Establishment of real-time fluorescence quantitative PCR (fluorescence RT-PCR) method

[0039] 1. Fluorescence RT-PCR reaction system

[0040] 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 22.0 μL, 2.5 mM dNTP 2.0 μL, 10 μM primer PH-F 1 μL, 10 μM primer PH-R 1 μL, 10 μM probe 1.5 μL, 5U / μL Taq DNA polymerase 0.3 μL, ddH 2 O 13.7 μL.

[0041] 2. Real-time fluorescent PCR reaction conditions

[0042] 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 which are used for detecting phytophthora hibernalis and a detection method for phytophthora hibernalis, the nucleotide sequence of the primer pair is shown as SEQ ID No. 1 and 2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. The invention further provides a RT-PCR method for detecting phytophthora hibernalis. The method comprises: taking total DNA of a sample as a template, utilizing the above primer pair and probe to perform real-time fluorescence PCR amplification, acquiring data after each circulation is finished, and determining the result according to an amplification curve after the reaction is finished. The method is capable of rapidly accurately detecting phytophthora hibernalis from in a complicated pathogenic bacteria environment in diseased plant tissues and soil. A detection kit constructed according to the method is simple in operation, good in specificity and high in sensitivity, and has important meaning on aspects of early warning of phytophthora hibernalis, pathogeny monitoring in epidemic areas, import and export safety and the like.

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 winteris and a detection method thereof. Background technique [0002] Phytophthora hibernalis Carne is one of the main pathogens causing brown rot of citrus. Citrus brown rot occurs before and after fruit harvest and can cause significant economic losses, especially in the rainy season. The pathogen was first discovered on citrus fruits in Western Australia, and was subsequently reported in many countries such as Europe, America and Africa. The fungus mainly infects the fruits and branches of citrus plants, and can also infect a variety of plants such as rhododendron, rose, safflower, sesame, tomato, apple, etc., resulting in wilting, root rot or ulcer symptoms. [0003] Therefore, the development of early and rapid detection technology for Phytophthora winteris is of great significance to the prevention and contr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/645
CPCC12Q1/04C12Q1/686C12Q2563/107
Inventor 杜洪忠吴品珊
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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