LAMP (loop-mediated isothermal amplification) detection primer composition, LAMP detection kit and LAMP detection method for P.tentaculata

A technology for detection kits and detection primers, which is applied in the field of LAMP detection primer compositions for Phytophthora woody, can solve the problems of long period, low sensitivity, and poor specificity of detection methods, and achieve high accuracy, increased application value, and practical good sex effect

Active Publication Date: 2015-02-25
瑞测精准医学检测(上海)有限公司
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Problems solved by technology

[0005] Purpose of the invention: Aiming at the problems of long period, poor specificity and low sensitivity of the biological detection method of Phytophthora xylini in the prior art, the purpose of the present invention is to provide a LAMP detection primer composition for Phytophthora xylini

Method used

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  • LAMP (loop-mediated isothermal amplification) detection primer composition, LAMP detection kit and LAMP detection method for P.tentaculata
  • LAMP (loop-mediated isothermal amplification) detection primer composition, LAMP detection kit and LAMP detection method for P.tentaculata
  • LAMP (loop-mediated isothermal amplification) detection primer composition, LAMP detection kit and LAMP detection method for P.tentaculata

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Experimental program
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Effect test

Embodiment 1

[0040] A LAMP detection kit for detecting P.tentaculata, the kit preferably includes 1ml detection solution, each milliliter includes: 32mM forward inner primer FIP, 32mM reverse inner primer BIP, 8mM forward outer primer F3, 8mM reverse outer primer B3, 8mM loop primer LB, 56mM dNTPs, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4) 2 SO 4 , 0.24mM MgSO 4 , 4% Triton X-100, 320 units of Bst DNA polymerase, and 180 mM hydroxynaphthol blue were added to ultrapure water until the total volume was 1 mL. The specific sequences of each primer are as follows:

[0041] FIP: 5'-ATCGTACGGATTTTCTGAGCAAAGTAGATCCCGATTTCCATCAG-3';

[0042] BIP: 5′-TGGACGGCAAGACCATCAAGTCCGTTAGTTAAATAAATACCTCGA-3′;

[0043] F3: 5'-TGCCTTATAGGAATAGCGC-3';

[0044] B3: 5'-AGCATAAGTGAATTGACCCA-3';

[0045] LB: 5'-CTCCAGATTGTACGTCCTTCGT-3'.

[0046]Wherein, the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, the reverse outer primer B3 and the reverse loop primer LB can a...

Embodiment 2

[0047] The specificity test of embodiment 2P.tentaculata LAMP reaction

[0048] In order to verify the specificity of the LAMP method, 10 P. tentaculata strains, 12 other oomycetes, and 19 pathogenic fungi from Jiangsu, Yunnan, Shandong, and Fujian provinces were used as test materials. The LAMP detection results showed that 10 P. Tentaculata strains can be observed with a sky blue positive reaction or agarose gel electrophoresis with LAMP ladder-like bands, and the remaining 12 oomycetes and 19 pathogenic fungi have purple negative reactions or no agarose gel electrophoresis. Amplified bands appear. Select different species (Phytophthora parasitica; Phytophthora sojae; Phytophthora capsici; Phytophthora strawberry; Phytophthora infestans) and different genera (Pythium ultima; Fusarium equiseti; bacteria; Rhizoctonia solani; Verticillium dahliae) DNA as a template, take 1 μL DNA solution, add 23 μL of the detection solution prepared in Example 1 and 1 μL sterilized deionized ...

Embodiment 3

[0049] The sensitivity test of embodiment 3P.tentaculata LAMP reaction

[0050] In order to determine the sensitivity of the LAMP detection method, the extracted DNA of P. tentaculata was measured with a spectrophotometer (1 μg / μL), then diluted 10 times with DEPC water, and stored at -70°C as a template. Take 1 μL of the 10-fold diluted DNA dilution solution of each concentration as a template, add 23 μL of the detection solution prepared in Example 1 and 1 μL of sterilized deionized water for LAMP reaction, and the reaction program is: 64°C for 80 min. Take 2 μL of the amplified product and run it on a 2% agarose gel for electrophoresis. The results show that the LAMP method can detect the DNA of P. tentaculata with a concentration of 1 ng; the HNB color reaction shows that the sensitivity of the LAMP reaction also reaches 1 ng ( image 3 , Figure 4 ).

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) detection primer composition, an LAMP detection kit and an LAMP detection method for P.tentaculata. The LAMP detection primer composition consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3 and a reverse loop primer LB. The detection method disclosed by the invention is high in accuracy, strong in specificity, convenient to operate and good in practicability, can be used for quickly and conveniently detecting the P.tentaculata with high efficiency, high specificity and high sensitivity under an isothermal condition of 64 DEG C without complex instruments, can well satisfy field detection of the P.tentaculata, provides a new technical platform for the detection of the P.tentaculata, can be used for high-sensitivity quick detection of the P.tentaculata, and can also be used for early diagnosis of epidemic diseases and monitoring of germs in pathogenetic fields.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP detection primer composition of Phytophthora tentaculata, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] Phytophthora tentaculata (P. tentaculata) was first isolated in 1993 from chrysanthemums showing root and stem rot in German nurseries, and then the pathogen was also found on verbena and chrysanthemum. In 2007, in Kunming, Yunnan, my country, the pathogen was also isolated from Muxiang. This is the first report of P. tentaculata in my country. According to the analysis of existing data, the pathogen mainly causes root rot and stem rot of Compositae plants, which is very harmful to the production of horticultural crops. Yunnan has always been the main supplier of flowers and horticultural plants in my country. How to formulate effective control measures to prevent the migration of Phytophthora species to other areas alon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 戴婷婷郑小波吴小芹
Owner 瑞测精准医学检测(上海)有限公司
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