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Potato rot nematode rDNA-ITS sequence, specific detection primer and one-step dual PCR detection method

A method of detection, technology of potatoes, applied in the field of biology

Inactive Publication Date: 2006-04-26
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

There is no molecular detection method for D. destructor in my country so far. The present invention is a rapid and specific molecular detection method for D. destructor in sweet potato type II.

Method used

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  • Potato rot nematode rDNA-ITS sequence, specific detection primer and one-step dual PCR detection method
  • Potato rot nematode rDNA-ITS sequence, specific detection primer and one-step dual PCR detection method

Examples

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Effect test

Embodiment 1

[0033] Example 1: Amplification of rDNA-ITS of D. destructor potato that damages sweet potatoes

[0034] 1.1 Extraction of stem nematode DNA

[0035] Pick 10 stem nematodes that harm sweet potatoes by hand, put them into a centrifuge tube filled with 14 μl sterilized redistilled water, and freeze them in a -20°C refrigerator for 2 hours. Rotate a glass rod sterilized with 75% alcohol in a centrifuge tube until the ice melts, add 3 μl of 10×PCR buffer, 3 μl of proteinase K solution (600 μg / ml), and freeze at -20°C for at least 1 hour. Take the centrifuge tube out of the refrigerator and incubate at 65°C for 1 hour to degrade deoxyribonucleic acid; then denature proteinase K at 95°C for 10 minutes, centrifuge at 10,000r / min for 1 minute, and take the supernatant DNA suspension in Store at -20°C for later use.

[0036] 1.2 PCR amplification of rDNA-ITS

[0037] The universal primers TW81 and AB28 designed by Joyce et al (1994) were synthesized by Shanghai Boya Biological Co., ...

Embodiment 2

[0099] Example 2: One-step double PCR detection method of D. destructor specific marker primers and universal primers (D3A and D3B)

[0100] The present invention adopts a one-step double PCR method, combines the above-mentioned specific primers SSsj and AB28 to amplify the specific fragment of D. destructor potato, and at the same time, in order to prevent false negative reactions, a general-purpose gene of the D3 region of the ribosomal gene 28S is added to the PCR reaction system. Primers D3A and D3B were used as internal standards, and the one-step double PCR method was used to detect D. destructor potato, which is harmful to sweet potatoes. The one-step double PCR of the same sample was repeated at least four times to confirm the research results and increase reliability.

[0101] The sequences of the specific primer SSsj, the universal primer AB28 and the ribosomal DNA28S gene D3 extension region universal primers D3A and D3B of D. destructor are as follows:

[0102] Ups...

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Abstract

The present invention is potato rot stem nematode rDNA-ITS sequence, specific detection primer and one-step double PCR detection method. The potato rot stem nematode rDNA-ITS has the nucleotide sequence as shown in SEQ No. 1 or SEQ No. 2. The specific detection primer SSsj designed based on the rDNA-ITS sequence and general purpose primer AB28 are combined and added with internal standard D3A and D3B for one-step double PCR detection. The PCR amplification result of potato stem nematode can obtain specific 623 bp segment and 345 bp segment, but that of other stem nematode can obtain specific 345 bp segment only. The present invention provides fast molecular detection method for stem nematode detection and identification and thus provides theoretic reference for comprehensive control of stem nematode disease.

Description

Technical field: [0001] The invention belongs to the field of biological technology, and relates to the rDNA-ITS sequence of D. destructor potato, a specific primer sequence designed for the sequence and a rapid PCR detection method for D. destructor potato by using the primer. technical background [0002] D.potato rot nematode (D.destructor) which damages sweet potato and D. dipsaci which damages garlic, corms and bulbs are important diseases of crops in my country. Sweet potato stem nematode disease has been reported in 21 counties and cities including Lulong County, Changli, Luan County, Qian'an, Langfang, Bazhou, Quyang County and Dingzhou in Hebei, Miyun, Daxing, Fangshan in Beijing, Xuchang in Henan, Zhengzhou, Pingdingshan, Jia County, Jilin City, Jilin City, Liaoning, Shandong Yishui County, Jiangsu Xuzhou and other provinces and cities are common, and the disease is very serious, mainly infecting the underground roots, tubers and tubers of sweet potatoes, and the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12Q1/68
Inventor 彭德良杨玉文
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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