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Phytopythiumvexans detection primers, LAMP detection system, kit and method

A detection kit and technology for Pythium spp., applied in the field of detection primers for Pythium spp., can solve the problems that Pythium spp. has not been reported, and there are few plant pathogenic oomycetes, so as to reduce blind use, facilitate operation, and reduce environmental pollution. pollution effect

Pending Publication Date: 2020-12-29
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this technology has been developed for nearly 20 years, there are few reports on the detection of plant pathogenic oomycetes, and there are no reports on the detection of Pythium belli at home and abroad.

Method used

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  • Phytopythiumvexans detection primers, LAMP detection system, kit and method
  • Phytopythiumvexans detection primers, LAMP detection system, kit and method
  • Phytopythiumvexans detection primers, LAMP detection system, kit and method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Pythium belli LAMP detection kit:

[0053] Contains 25 μL detection solution, detection solution includes: 1.6 μm forward inner primer FIP, 1.6 μm reverse inner primer BIP, 0.2 μm forward outer primer F3, 0.2 μm reverse outer primer B3, 0.8 μm loop primer LF, 1.4 mM dNTPs , 1×LAMPBuffer, 1×xylenol orange, 0.8M betaine, 8mM MgS04, 8U Bst DNA polymerase, 1μL DNA template, sterilized water to make up to 25μL.

[0054] Wherein, each primer sequence is specifically as follows:

[0055] FIP: 5'-GTGTAAACTAAAAATAGCTAACTTATCCACCATTATCAAGTGTAG-3';

[0056] BIP: 5'-CGCAATTAATTTTATTGTTACTAGCATTCTATGCATACTTAATCCA-3';

[0057] F3: 5'-ATCAGGTGCAGGTACTGG-3';

[0058] B3: 5'-AGCTGTAATTAAAAAGAGACCTATC-3';

[0059] LF: 5'-TGAAGGTCCTGAGTGTGCAG-3'.

[0060] Wherein, forward inner primer FIP, reverse inner primer BIP, forward outer primer F3, reverse outer primer B3 and forward loop primer loop primer LF can directly form the detection primer for detecting Pythium belli.

Embodiment 2

[0061] Embodiment 2: Specificity test of Pythium belli LAMP reaction

[0062]In order to verify the specific primer sequence of Pythium belli, utilize the detection kit among the embodiment 1, take 3 Pythium belli strains and 18 kinds of other bacterial strains as test material (Table 1), LAMP detection result shows 3 strains All Pythium bellii strains could observe yellow color change, and LAMP ladder-like bands could be observed after electrophoresis. Select different species (Phytopythium chamaehyphon, P.montanum, P.boreale, P.citrinum, P. helicoides, P.oedochilum) and different genera (Pythium myriotylum, Py.intermedium, Py.ultimum, Py. Heterothallicum, Py.irregulare, Phytophthorasyringae, Phy.cinnamomi, Phy.infestans, Phy.cambivora, Phy.cactorum, Verticilliumalboatrum, Fusariumoxysporum) DNA as a template, take 1μL DNA solution, add 24μL detection solution for LAMP reaction, the reaction procedure is 65°C for 60min.

[0063] Table 1 Specific fungal and oomycete strains ...

Embodiment 3

[0067] Embodiment 3: LAMP reaction sensitivity test of Pythium belli

[0068] In order to determine the sensitivity of the LAMP detection method, the DNA concentration of the extracted Pythium belligerium was measured with Qbuit 3.0 (Thermo Fisher Scientific) and adjusted to 1 ng / μL, and then diluted 10 times with ultrapure water. Take 1 μL of 10-fold diluted DNA dilutions of each concentration as a template, add 24 μL of detection solution to carry out LAMP reaction, and the reaction program is 65 ° C for 60 min. Take 8 μL of the amplification product and load it;

[0069] For specific test results, see figure 2 , where M is a 100bp DNA marker; in the figure 2(b) color judgment LAMP detection Pythium bell sensitivity color chart; the figure shows that the 1st, 2nd, 3rd and 4th tubes are yellow and positive; the 5th tube is purple Negative, the No. 6 negative control is purple; 25μL reaction system contains 1ng, 100pg, 10pg, 1pg, 0.1pg of Pythium bellia, which contains 1ng,...

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Abstract

The invention discloses Phytopythiumvexans detection primers, LAMP detection system, kit and method. The detection primers include a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3, a backward outer primer B3 and a forward loop primer LF. Compared with a traditional detection technology for identifying Phytopythiumvexans according to morphological characteristics,the detection method provided by the invention has the advantages of high accuracy and sensitivity and convenience in operation, and provides a new technical platform for Phytopythiumvexans detectionwhile realizing constant-temperature amplification. The method can be used for high-sensitivity rapid detection of the Phytopythiumvexans in production practice, pathogens can be identified in the early stage of disease infection, and the Phytopythiumvexans in field soil can be detected. According to the invention, reliable technical and theoretical bases are provided for prevention and treatmentof the Phytopythiumvexans.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection primer for Pythium bellis, a LAMP detection system, a kit and a method. Background technique [0002] Phytopythiumvexans belongs to Chromista, Oomycota, Oomycetes, Pythiales, Pythiaceae, Phytopythium . Pythium bellis is a soil-borne pathogenic Pythium with a wide range of hosts. It can infect various economic crops and medicinal plants such as rubber trees, potatoes, sugarcane and kiwi, causing root diseases, seedling rot and other symptoms , and has a worldwide distribution and has become one of the important pathogenic bacteria of many crop seedling diseases. At present, there is no effective detection and control measures for this disease, so the establishment of rapid molecular detection of Pythium bellii is of great significance for the early control of the disease caused by it. [0003] Currently, there are few detection techniques for Pythium bellii....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6895C12Q1/6844C12Q2531/119
Inventor 李明珠高菡张鑫强毅崔浪军闫亚平
Owner SHAANXI NORMAL UNIV
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