Phytophthora nicotianae molecule detection primer and detection method thereof

A technology of Phytophthora nicotianae and a detection method, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of false positives, long required period, and low sensitivity of immunoserological identification methods, and achieve practical The effect of good performance, reliability guarantee and high sensitivity

Inactive Publication Date: 2014-02-05
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide Phytophthora nicotiana specific molecular detection primers and A method

Method used

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  • Phytophthora nicotianae molecule detection primer and detection method thereof
  • Phytophthora nicotianae molecule detection primer and detection method thereof
  • Phytophthora nicotianae molecule detection primer and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Design of primer sequences and specific amplification of the primers against Phytophthora nicotianae

[0035] 1. Extraction of genomic DNA of test strains

[0036] In order to obtain the specific primer sequence of Phytophthora nicotianae, 31 strains of Phytophthora nicotinicum and several related species of Phytophthora spp. and several common pathogenic fungi from Fujian, Yunnan and Taiwan provinces of China were used as test materials, and the CTAB method was used to extract The specific method for the genomic DNA of the tested strain is as follows: Take 50mg of freeze-dried mycelium powder in a 1.5ml centrifuge tube, and add 900μl of 2% CTAB (hexadecyltrimethylammonium bromide) extract (2% CTAB; 100 m mol / L Tris-HCl, PH 8.0; 20mmol / L EDTA, pH 8.0; 1.4 mol / L NaCl) and 90 μl 10% SDS (sodium dodecylbenzene sulfonate), then mix well, at 55~ 60℃ water bath for 1.5 h, shake and mix once every 10 min. After 1.5 h in water bath, centrifuge (12,000 rpm) for 15 min. T...

Embodiment 2

[0045] Example 2: Detection of sensitivity of primers to Phytophthora tabaci

[0046] 1. DNA concentration dilution: The extracted genomic DNA of Phytophthora nicotianae is diluted in series after the concentration is measured by a spectrophotometer.

[0047] 2. Sensitivity Detection of Phytophthora Nicotiana

[0048] First, use YPT1 universal primer (ph1F: 5'-CGACCATTGGCGTGGACTTT-3',

[0049] Yph2R: 5'-ACGTTCTCGCAGGCGTATCT-3') was used as the first round reaction primer, and PCR amplification was carried out according to the following reaction system and reaction procedure. 25 μl PCR reaction system, including 2.5 μL 10×PCR buffer (Mg 2+ free), 2.0 mmol / L MgC1 2 , 0.2 mmol / L dNTP, 1.0 U Taq DNA polymerase (Takara Dalian Bao Bioengineering Co., Ltd.), primers (ph1F / Yph2R) 0.4 μmol / L each, 25 ng DNA template, the insufficient part is made up by sterile ultrapure water. The amplification program is: 95 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s; 58 ℃ annealing for...

Embodiment 3

[0052] Example 3: Detection of Phytophthora nicotianae in diseased plant tissues

[0053] 1. Sample collection: Plant tissue samples were collected from the tobacco base in Nanping City, Fujian Province.

[0054] 2. DNA extraction and detection

[0055] NaOH rapid lysis method is used to extract DNA from diseased plant tissues. The specific process is as follows: ①Wash and dry tobacco diseased leaves or stems, and cut the diseased parts; ②Add 10uL (0.5 mol / L) to 1 mg diseased leaves or stems. NaOH, 0.5% PVP), the tissue is fully pulverized into a paste, and centrifuged in a 12,000 g centrifuge for 6 min; ③ Take 20 uL of the supernatant and mix with an equal volume of 0.1 mol / L Tris-HCl (pH 8.0); ④ Take 1 uL as a PCR template for amplification.

[0056] PCR amplification reaction system 25 μl, including 2.5 μL 10×PCR buffer (Mg 2+ free), 2.0 mmol / L MgC1 2 , 0.2 mmol / L dNTP, 1.0 U Taq DNA polymerase (Takara Dalian Bao Biological Engineering Co., Ltd.), primers (YhF / YhR) 0.4 μmol...

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Abstract

The invention discloses a Phytophthora nicotianae molecule detection primer and a detection method thereof, specially used for the specific molecule detection of Phytophthora nicotianae. A pair of specific primers of the Phytophthora nicotianae (comprising an upstream primer YhF:5'-GACATGATATCAACTGTTCTGCAG-3' and a downstream primer YhR:5'-CCTTGGATCTTCTCTCGATAAG-3') are mainly designed, and an amplified product having a fragment length of 342bp can be specially amplified on the pure DNA and bacteria bearing pathogenic tissues of the Phytophthora nicotianae through PCR amplification and agarose gel electrophoresis. The primer and the method can be used for the rapid, sensitive and specific detection of the Phytophthora nicotianae in Phytophthora nicotianae infected plant tissues in the production practices, can be used for the early stage diagnosis of field diseases and the monitoring and identification of pathogens, and provide reliable technologic and theoretic bases for the control of diseases caused by the Phytophthora nicotianae.

Description

technical field [0001] The invention relates to a primer for molecular detection of Phytophthora tobacco and its detection method, which is specially used for high-sensitivity and rapid molecular detection of Phytophthora tobacco, and can be used for early diagnosis of tobacco black shank disease in the field and monitoring and identification of pathogens, belonging to crop disease detection, Identification and prevention technology field. Background technique [0002] by Phytophthora tabacum ( Phytophthora nicotianae Tobacco black shank caused by Breda de Haan is a devastating worldwide disease. In 1896, van Breda de Haan was first discovered in Java, Indonesia. Since then, the disease has spread rapidly. In 1922, it became a serious disease in Florida and Kansas in the United States. After 1924, the disease has spread all over the tobacco fields in temperate, subtropical and tropical regions of the world. It was first reported in China in 1950. At that time, the disease...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2531/113
Inventor 李本金陈庆河翁启勇何美玲谢世勇谢廷鑫
Owner INST OF PLANT PROTECTION FAAS
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