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RPA detection-based quarantine solanum weed solanum elaeagnifolium detection method

A silver-haired black nightshade and DNA molecular technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long time, time-consuming, laborious, and long-term, and achieve high specificity and Stability, simple and fast response, and short response time

Inactive Publication Date: 2016-03-16
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These technologies are mainly PCR-based variable temperature amplification technology, in which RAPD map stability is poor, and RFLP technology is time-consuming and labor-intensive, and several existing barcode gene mutation sites of the four species of Solanum quarantine weeds are fixed, and RFLP is used to distinguish more difficult
At present, the widely used DNA barcoding technology uses general primers for specific genes to amplify and sequence the obtained fragments. It takes a long time, and it takes longer for remote quarantine departments. The evolution is relatively slow, the variation is small, the similarity is above 92%, and it is difficult to identify the species

Method used

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  • RPA detection-based quarantine solanum weed solanum elaeagnifolium detection method
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  • RPA detection-based quarantine solanum weed solanum elaeagnifolium detection method

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, the establishment of the preparation of the RPA kit of identification silver-haired nightshade and identification method

[0039] 1. Design of RPA primers for identification of Solanum nigrum

[0040] The present invention is used for the identification kit of the RPA of black nightshade, which contains the primer pair for specific detection of black nightshade, and the recombinase, single-stranded DNA binding enzyme, strand-displacing DNA polymerase, dNTPs, Magnesium acetate, etc. The primer pair design process is roughly as follows:

[0041] According to the nuclear gene Solanumelaeagnifolium voucher Bohs3204UT granule-boundstarch synthase (GBSSI) gene (NCBI: AY996412.1), a quarantine solanum weed, multiple sets of theoretically feasible RPA primers were designed using primer design software, and two sets of primers were selected for specificity experiments Verify that the two sets of primers are:

[0042] Waxy-F1: 5'-AGCTTGTTCTGTCAAGTAAGTTACTAGCTGTAT...

Embodiment 2

[0069] Example 2, Rapid Analysis of RPA

[0070] With the leaves and stems of Solanum nigrum as the test sample, the primer pair (Waxy-F1 / Waxy-R1, sequence 1 and sequence 2) screened and identified in the above-mentioned embodiment 1 is used for RPA detection, and several reaction time gradients are designed, i.e. 10min , 20min, 30min, 40min, 60min, 90min to analyze the influence of RPA reaction time on detection. Refer to Step 2 of Example 1 for the rest of the operations.

[0071] It was found that after 10 minutes of reaction, the target band with a size of 210bp (sequence 3) could be detected. After 20 minutes, the brightness of the amplified fragment did not increase significantly, and after 90 minutes of reaction, the brightness decreased, indicating that the RPA reaction can be performed in a relatively short time. The DNA fragments were amplified within 20 minutes, and this experiment determined that 20 minutes was the optimal time for the RPA reaction. experiment as...

Embodiment 3

[0072] Embodiment 3, the sensitivity analysis of RPA detection

[0073] The total DNA of the quarantine Solanum weed Solanum aureus was diluted from 20.8ng / μL to 5.2, 1.04, 0.52, 0.104, 0.052, 0.0104ng / μL for RPA detection. In a 25 μL reaction system, 1 μL of the above solution was added to obtain 5.2, 1.04, 0.52, 0.104, 0.052, and 0.0104 ng of DNA templates. Refer to Step 2 of Example 1 for other operations.

[0074] The results of the RPA reaction showed that when the DNA template was 0.0104ng, the target fragment (210bp, sequence 3) was relatively clear, which proved that the RPA reaction had higher sensitivity. Experimental results such as image 3 shown.

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Abstract

The invention discloses a RPA detection-based quarantine solanum weed solanum elaeagnifolium detection method. A primer pair used for identifying solanum elaeagnifolium is provided, and is a primer pair selected from: (a) a primer pair composed of two single-stranded DNA molecules represented by a sequence 1 and a sequence 2 respectively; or (b) a primer pair composed of two single-stranded DNA molecules represented by the sequence 1 and sequence 2 after substitution and / or deletion and / or addition of one or a plurality of nucleotides; wherein the function of the primer pair disclosed in (a) and the function of the primer pair disclosed in (b) are the same. The primer pair is used for RPA detection of quarantine weed solanum elaeagnifolium, and is extremely high in specificity and sensitivity; operation is convenient; detection time is short; and solanum elaeagnifolium can be distinguished from three expressly stated quarantine solanum weeds including Solanum carolinense, Solanum rostratum, and Solanum torvum effectively via application of the primer pair.

Description

technical field [0001] The invention belongs to the field of plant quarantine and relates to an RPA-based method for detecting the quarantine Solanum solanum weed. Background technique [0002] There are more than 1,400 species of Solanum in the world, most of which originated in South America, are widely distributed, have strong adaptability, and are very harmful. (Solanumtorvum) was included in the "List of Imported Plant Quarantine Pests of the People's Republic of my country" promulgated by China in 2007, and there are more than 30 species that are potentially harmful. Molecular phylogeny studies of the genus Solanum show that Solanum nigrum belongs to the subgenus Leptostemonum, which has 350-450 species, and belongs to 10 subclades on the evolutionary tree. Almost all the fruits and seeds were intercepted and found at the port. The seeds and fruits of several quarantine weeds of the genus Solanum and their related species are very similar in shape, especially the ident...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2521/101C12Q2522/101
Inventor 雷荣蒋弘山胡帆范晓红
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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