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Soy bean cyst roundworm specific SCAR label, specific primer and quick PCR detecting method

A kind of soybean cyst nematode, detection method technology, applied in biological field

Inactive Publication Date: 2007-01-24
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although there are reports of soybean cyst nematode-specific primer GlyF1 based on the ITS region of ribosomal DNA at home and abroad, there are no reports of soybean cyst nematode-specific SCAR marker primers based on genomic DNA at home and abroad to detect soybean cyst nematodes

Method used

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  • Soy bean cyst roundworm specific SCAR label, specific primer and quick PCR detecting method
  • Soy bean cyst roundworm specific SCAR label, specific primer and quick PCR detecting method
  • Soy bean cyst roundworm specific SCAR label, specific primer and quick PCR detecting method

Examples

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Effect test

Embodiment 1

[0035] Example 1: DNA fragments and specific primers for soybean cyst nematode-specific SCAR markers

[0036] 1.1 Extraction of soybean cyst nematode DNA

[0037] One cyst of each group of soybean cyst nematode, white nematode potato, and golden nematode potato was picked by hand, put into 14 μl sterilized redistilled water, and frozen in a -20°C refrigerator for 1 hour. Rotate the glass rod sterilized with 75% alcohol in an eppendorf tube until the ice melts, add 3 μl of 10X PCR buffer, 3 μl of proteinase K solution (600 μg / ml), and freeze at -20°C for at least 2 hours. After 2h, take the eppendorftube out of the refrigerator and incubate at 65°C for 1.5h to degrade deoxyribonucleic acid; then denature proteinase K at 95°C for 10min, centrifuge at 1000rpm for 1min, and take the supernatant DNA suspension in - Store at 20°C for later use.

[0038] 1.2 Screening of soybean cyst nematode-specific RAPD markers (SCAR markers)

[0039] According to Peng Deliang (Peng Deliang, wh...

Embodiment 2

[0057] Example 2: Molecular detection of soybean cyst nematode-specific SCAR marker primers

[0058] The soybean cyst nematode-specific SCAR marker primers SCNFI and SCNRI constructed by the present invention were synthesized by Shanghai Boya Biological Co., Ltd. The total volume of the specific SCAR marker amplification reaction system was 50 μl, which contained 5 μl 10XPCRbuffer (containing Mg++); 4 μl 2.5mM dNTP; 0.5μl SCNFI (0.1μg / μl); 0.5μl SCNRI (0.1μg / μl); 1UTaq DNA polymerase (5U / μl); 2μl template DNA (extracted from soybean cyst nematode and other cyst nematode populations DNA); ddH 2O 37.8 μl.

[0059] The applied soybean cyst nematode specific SCAR marker primer sequence is as follows:

[0060] SCNFI (5'-GGA CCC TGA CCA AAA AGT TTC CGC-3'

[0061] SCNRI (5'-GGA CCC TGA CGA GTT ATG GGC CCG-3'

[0062] The PCR amplification program was as follows: pre-denaturation at 94°C for 4min, followed by 35 cycles of 94°C for 30S, 75°C for 60S, 72°C for 2min, extension at 72...

Embodiment 3

[0064] Example 3: Specific SCAR marker primers and universal primers (D2A and D3B) one-step double PGR method to detect soybean cysts

[0065] Nematodes

[0066] In the present invention, the constructed soybean cyst nematode-specific SCAR marker primers SCNFI, SCNRI and universal primers D2A and D3B are placed in the same PCR reaction, and SCNFI and SCNRI specifically amplify the SCAR of soybean cyst nematode genomic DNA To mark the fragment, primers D2A and D3B were used to amplify the DNA fragment in the extended region of rDNA gene D2 and D3. The one-step double PCR method adopted in the present invention is the world's first soybean cyst nematode molecular diagnosis method, and the molecular detection method contains two fragments of genomic DNA and ribosomal DNA.

[0067] The soybean cyst nematode molecular detection method of the present invention contains a PCR reaction system of 20 μl, and the proportion is as follows: 10XPCR-buffer (containing Mg++) 2.0 μl, dNTPs 1....

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Abstract

The present invention is specific SCAR label and specific primer of soybean cyst roundworm and their quick PCR detection method, and belongs to the field of biotechnology. The specific SCAR label of soybean cyst roundworm features its nucleotide sequence as shown in SEQ No. 1. The specific primers SCNF1 and SCNR1 designed based on the specific SCAR label of soybean cyst roundworm may be used in the quick PCR detection of soybean cyst roundworm and can amplify a 500 bp segment. It is also possible to add primers D2A and D3B as internal standards to amplify a 800 bp segment simultaneously. The present invention has raised molecular detection sensitivity, fast detection speed, accurate detection result and very high application value in the early detection of soybean cyst roundworm and the early diagnosis of soybean cyst roundworm disease.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a soybean cyst nematode-specific SCAR marker complete sequence, a specific SCAR marker primer sequence and a soybean cyst nematode SACR-specific rapid PCR detection method. technical background [0002] Soybean cyst nematode (Heterodera glycines) is a major destructive biological disaster in soybean production in the world. It occurs widely in the United States, Japan, South Korea, China, the Russian Far East, Argentina, Brazil, Egypt, Indonesia and other countries, causing serious damage. In my country, soybean cyst nematodes are mainly distributed in the two main soybean producing areas of the Northeast and Huanghuaihai, especially in the west of the three northeastern provinces where sandstorms, drought, and saline-alkali land are generally serious. The disease causes a loss of 10%-20%, and when it is serious, it reaches 30%-50%, which reduces soybean production by about 400 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C07H21/00C12Q1/68
Inventor 彭德良欧师琪
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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