Singularity SCAR mark of wheat heterodera avenae sensu lato and rapid PCR detection method thereof

A cereal cyst nematode, specific technology, applied in the field of quick PCR detection of wheat cereal cyst nematode SACR specific markers, to achieve the effect of strong specificity, good accuracy and high sensitivity

Inactive Publication Date: 2012-07-11
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

Mainly focused on ITS sequence characteristics, using restriction enzymes to analyze the length polymorphism of ITS fragments, but there is no report on the specific detection of wheat cyst nematode based on genomic DNA-based SCAR marker primers

Method used

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  • Singularity SCAR mark of wheat heterodera avenae sensu lato and rapid PCR detection method thereof
  • Singularity SCAR mark of wheat heterodera avenae sensu lato and rapid PCR detection method thereof
  • Singularity SCAR mark of wheat heterodera avenae sensu lato and rapid PCR detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: DNA fragments, specific primers of wheat cereal cyst nematode-specific SCAR markers

[0037] 1.1 Extraction of genomic DNA of wheat cereal cyst nematode

[0038] Pick one full cyst of cyst nematodes such as wheat cereal cyst nematode, Philip cyst nematode, soybean cyst nematode upland rice cyst nematode, barley cyst nematode, etc., or single root knot Larvae of nematodes such as nematode, D. destructor, pine wood nematode, and banana borer nematode were put into PCR tubes containing 10 μl of double-distilled water, and quickly frozen in liquid nitrogen. For root-knot nematodes, nematode populations such as Dilomata sweetpotato, single-headed nematodes were picked and put into 10 μl of sterilized double-distilled water, which was also frozen in liquid nitrogen. After taking it out, place it on ice, turn a glass rod sterilized with 75% alcohol in the PCR tube until the ice melts, and pierce the cysts to release the eggs therein. Add 8 μl of 10× PCR buffer,...

Embodiment 2

[0053] Example 2: Molecular detection of wheat cereal cyst nematode-specific SCAR marker primers

[0054]Wheat cereal cyst nematode-specific SCAR marker primers HaF1 and HaR1 constructed in the present invention were synthesized by Shanghai Sangon Biology Co., Ltd. The total volume of the amplification reaction system for specific SCAR markers was 50 μl, which contained 5 μl 10×PCR buffer (including Mg++); 4μl dNTP; 1.0μl HaF1 (0.1μg / μl); 1.0μl HaR1 (0.1μg / μl); 2.5U Taq DNA polymerase (5U / μl); 2μl template DNA (wheat cereal cyst nematode and other Genomic DNA extracted from cyst nematode populations); ddH 2 O 36.5 μl.

[0055] The applied wheat cereal cyst nematode specific SCAR marker primer sequence is as follows:

[0056] HaF1: 5'-TGACGAGAACATATGATGGGGAT-3'

[0057] HaR1: 5'-GAGGGGGTGGGAATGAAATGGAT-3'

[0058] The PCR amplification program was as follows: pre-denaturation at 94°C for 5min, followed by 35 cycles of 94°C for 30S, 59°C for 30S, 72°C for 2min, extension at ...

Embodiment 3

[0061] Embodiment 3: Sensitivity determination of wheat cereal cyst nematode SCAR marker detection

[0062] The genomic DNA of cereal cyst nematode in Qingyundian area of ​​Beijing was used as the initial template, and after gradient dilution, these gradiently diluted genomic DNA were amplified with SCAR primers. When the template concentration was greater than 10 -2 A specific 1010bp band ( image 3 ).

[0063] The PCR reaction system is 25 μl, and the ratio is as follows: 10×PCR-buffer 2.5 μl; 10 mmol / L dNTP 2.0 μl; primer HaF1 (0.1 μg / μl) 1.0 μl; HaR1 (0.1 μg / μl) 1.0 μl; Taq DNA polymerase 0.25 μl (5U / μl); template DNA 1.0μl; sterilized redistilled water to supplement 25μl.

[0064] The amplification program of the PCR reaction was as follows: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30S, 59°C for 30S, and 72°C for 2 minutes, and after extension at 72°C for 10 minutes, store at 4°C. Take 5 μl of amplified product and add 1 μl loading buff...

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Abstract

The present invention provides a singularity SCAR mark of a wheat heterodera avenae sensu lato and a rapid PCR detection method thereof, which belongs to the field of biotechnology. DNA fragments of the SCAR mark of the wheat heterodera avenae sensu lato is characterized in that the DNA fragments are provided with a nucleotide sequence shown as SEQ ID NO 1. Auele specific primers HaF1 and HaR1 designed according with the DNA fragments of the singularity SCAR mark of the wheat heterodera avenae sensu lato can amplify a singularity fragment with 1010bp in the PCR rapid detection method for the wheat heterodera avenae sensu lato. According to the Singularity SCAR mark and the detection method, the molecule detection sensitivity is increased and the detection is rapid and accurate, which has a high application valve in the detection aspect of the wheat heterodera avenae sensu lato and the early diagnosis aspect of wheat heterodera glycine.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a complete sequence of wheat cereal cyst nematode specific SCAR markers, a specific SCAR marker primer sequence and a rapid PCR detection method for wheat cereal cyst nematode SACR specific markers. Background technique [0002] Wheat cereal cyst nematode (Heterodera avenae; commonly known as Cereal Cyst Nematode, CCN) is a worldwide important pathogenic nematode on temperate cereal crops. More than 40 countries including China, the United States, and China have suffered damage to varying degrees, seriously affecting the yield and quality of cereal crops, especially wheat crops. In my country, since Peng Deliang and others first discovered that wheat cereal cyst nematodes caused damage to wheat in Tianmen County, Hubei Province in 1989, it has been found in Hubei, Henan, Hebei, Beijing, Shandong, Shanxi, Inner Mongolia, Qinghai, Jiangsu, Anhui, Gansu, and Shaanxi. Cereal cyst n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 彭德良亓晓莉黄文坤彭焕贺文婷
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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