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Prunus salicina acalitus phloeocoptes PCR detection primer and detection method thereof

A detection method and a technology for detecting primers, which are applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problems of low sensitivity of microscopic examination of P. , to achieve high sensitivity, accurate and reliable detection results, and guaranteed reliability

Active Publication Date: 2016-11-02
POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the problems of low microscopic examination sensitivity and high professional quality requirements of test personnel in the existing detection technology, and to provide specific PCR detection primers and reliable detection results, high sensitivity and specificity Robust and easy-to-operate PCR method for the detection of P.

Method used

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  • Prunus salicina acalitus phloeocoptes PCR detection primer and detection method thereof
  • Prunus salicina acalitus phloeocoptes PCR detection primer and detection method thereof
  • Prunus salicina acalitus phloeocoptes PCR detection primer and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Specific amplification of PCR primers to the phyllophyll mite

[0029] 1. Extraction of template DNA

[0030] In order to obtain the specific primers for P. oleifera, the total DNA of flower buds was extracted by the CTAB method using 21 P. oleifera and a number of closely related mites in Ningde, Nanping and Longyan, Fujian as test materials. The specific steps As follows: Take 0.5 g of the damaged flower bud tissue, put it in a mortar, grind it into a fine powder with liquid nitrogen; add 900 μL 2% CTAB, 10 μL β-mercaptoethanol, 90 μL 10% SDS, 2 μL proteinase K; 65 ° C, water bath for 1 hour, cool to room temperature, centrifuge at 12000rpm for 10min; take the supernatant, add an equal volume of atmosphere / chloroform / isoamyl alcohol (25:24:1), invert gently, let stand for 5min, and centrifuge at 12000rpm for 10min; take the supernatant, add an equal volume of chloroform / isoamyl alcohol Isoamyl alcohol (24:1), gently inverted, let stand for 5 minutes, centr...

Embodiment 2

[0041] Example 2: Sensitivity detection of PCR primers to Prunus chinensis

[0042] 1. Dilution of template total DNA concentration

[0043] The DNA concentration was determined by a nano-volume spectrophotometer (NanoDrop), and the extracted total DNA was diluted into 7 different concentrations of 50ng, 5ng, 500 pg, 50 pg, 5 pg, 500 fg and 50 fg by a 10-fold concentration serial dilution method gradient.

[0044] 2. Sensitivity detection

[0045] Take 1 μL of total DNA as a template for PCR detection. The PCR reaction conditions are as follows:

[0046] 25 μL PCR reaction system: including 12.5 μL PCR Mix, 1 μL each of 10 μM YM-F3 and 10 μM YM-B3, 50 ng of total DNA template, made up to 25 μL with sterilized double distilled water. The amplification program was pre-denaturation at 94°C for 4 min, deformation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 32 cycles, and finally extension at 72°C for 7 min.

[0047] 3. Test results

[0048] For ...

Embodiment 3

[0049] Example 3: Field measurement of the damage of P. prune flower buds by the hair mite.

[0050] 1. Sample collection: Samples were collected from Ningde, Nanping and Longyan oil and gas production bases in Fujian.

[0051] 2. DNA extraction and detection

[0052] The total DNA of the harmed oily flower buds was extracted by CTAB method.

[0053] Perform PCR testing as follows:

[0054] ①PCR reaction system 25 μL: including 12.5 μL PCR Mix, 1 μL each of 10 μM YM-F3 and 10 μM YM-B3, 50 ng of total DNA template, made up to 25 μL with sterilized double distilled water.

[0055] ②The amplification program was pre-denaturation at 94°C for 4 minutes, deformation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 32 cycles, and finally extension at 72°C for 7 min.

[0056] 3. Test results

[0057] For sample test results, see image 3 . A 214bp specific band appeared in the amplified product after agarose gel electrophoresis, which indicated that the ...

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Abstract

The invention discloses a prunus salicina acalitus phloeocoptes PCR detection primer and a detection method thereof and belongs to the technical fields of insect pest detection, identification, prevention and treatment of fruit trees. A prunus salicina acalitus phloeocoptes PCR detection primer is designed in the invention, wherein the sequence is represented as SEQ ID No.1-2. After a PCR reaction, agarose gel electrophoresis detection is carried out and a target electrophoresis band can be observed. The PCR detection primer and the detection method can be used for detection of the acalitus phloeocoptes in flower buds of the prunus salicina and also can be used in early-stage detection, monitoring and identification of the prunus salicina acalitus phloeocoptes in the field. The invention provides reliable technical and theoretical basis for prevention and treatment on the insect pests of the prunus salicina due to the acalitus phloeocoptes. The method, compared with a conventional method, is simple and quick, saves time and labor, is easy to learn for non-professional classification operators, and belongs to a molecular biological method of quickly identifying the acalitus phloeocoptes.

Description

technical field [0001] The invention relates to a PCR detection primer and a detection method thereof, which are specially used for the high-sensitivity and rapid molecular detection of the hairy gall mite, and can be used for the early diagnosis, monitoring and identification of the hairy mite in the field, and belong to fruit trees The technical field of pest detection, identification and control. Background technique [0002] Oil ( Prunus salicina Lindli var cordata J.Y.Zhang et al), also known as 㮈李, belongs to Rosaceae ( Rosaceae ) Prunoideae (Prunoideae) Prunus ( Prunus L) Plant is a good fruit produced in Fujian, and it is also the most distinctive branch industry in my country's plum industry. In addition to Fujian, Guangdong, Guangxi, Hunan, Jiangxi and other provinces and regions have introduced species. It is an important cultivated tree species in southern fruit producing areas. It is of great significance to develop the rural economy and help fruit farmers ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 胡菡青范国成林雄杰王贤达罗水鑫陈瑾
Owner POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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