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Real-time fluorescent LAMP detection method and kit for Phytophthora infestans

A potato late blight and real-time fluorescence technology, applied in the field of molecular biology, can solve the problems of contamination of amplification products, time-consuming and labor-intensive, etc., and achieve the effects of high sensitivity, ensuring accuracy, and convenient and fast detection methods.

Inactive Publication Date: 2018-10-09
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of LAMP can be determined by visually observing whether there is white magnesium pyrophosphate precipitation, but it is easy to cause misjudgment due to visual differences; adding fluorescent dye SYBRGreen I for color development is likely to cause contamination of the amplification product, resulting in misjudgment; carry out 1.2 % agarose gel electrophoresis, observation of ladder-shaped bands is time-consuming and laborious

Method used

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  • Real-time fluorescent LAMP detection method and kit for Phytophthora infestans
  • Real-time fluorescent LAMP detection method and kit for Phytophthora infestans
  • Real-time fluorescent LAMP detection method and kit for Phytophthora infestans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Real-time fluorescent LAMP detection primer set for P. infestans

[0057] Homology analysis was performed on the Ypt1 gene sequence of P. infestans Ypt1 retrieved from Genebank (accession number DQ162961), and the species conservation region was determined. The primer design software primerexplorer V4 (https: / / primerexplorer.jp / elamp4.0.0 / index.html) was used to design LAMP primers, and 1000 pairs of primer sequences were obtained.

[0058] The presence or absence of primer dimers and neck ring structure analysis on the primers by DNAMAN, and through ordinary PCR and LAMPPCR experiments, 6 primers with strong specificity and high sensitivity were selected to form the primer set of the present invention, including:

[0059] Two outer primers:

[0060] F3: 5'-CAAGACCATCAAGCTCCAA-3';

[0061] B3: 5'-GTCCGTCACATCGTACAC-3';

[0062] Two inner primers:

[0063] FIP: 5'-TCACGCGGGGACAAATGTTGTACGCCGTACCATCAAG-3';

[0064] BIP: 5'-TTCAACAGTGGGACACTGCCGATAATACCGTGGG...

Embodiment 2

[0068] The detection of embodiment 2 potato infestation bacteria

[0069] 1. Collection of Phytophthora infestans Bacteria and Extraction of Genomic DNA

[0070] The Phytophthora infestans strain was cultured on rye medium, and the hyphae were collected after 10 days. Genomic DNA was extracted by the modified CTAB method. The obtained DNA was used as a LAMP template.

[0071] 2. 25 μL fluorescent modified LAMP reaction system

[0072] 25 μL fluorescence-modified LAMP reaction system: 25 μL reaction system, inner primer 1.2 μM, outer primer 0.32 μM, loop primer 0.68 μM, dNTPs 1.8 mM, MgSO 4 1.5mM, 8U / μL Bst DNA polymerase, 3.2μL, 10×ThermoPolReaction Buffer (New England Biolabs, MA) (20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO 4 , 2mM MgSO 4 , 0.1% Triton X-100) 2.5 μL, 8U / μL Bst DNA polymerase 3.2 μL, DNA template 2 μL, 50×SYBR Green Ⅰ 0.3 μL, ddH 2 O to make up to 25 μL.

[0073] 3. Judgment of amplification and detection results

[0074] ①The LAMP reaction syste...

Embodiment 3

[0084] Example 3 Optimization of real-time fluorescent LAMP reaction system

[0085] (1) Screening of optimum betaine concentration

[0086] Betaine (50mol·L -1 ) respectively set 7 different treatments such as 0μL, 0.5μL, 1.0μL, 1.5μL, 2.0μL, 2.5μL, 3.0μL, and the amplification curves are arranged from top to bottom according to the fluorescence value as follows Figure 4 As shown, the test results show that the earliest peak is without betaine, so it is determined that the optimum amount of betaine added is 0 μL.

[0087] (2) Optimum Mg 2+ concentration screening

[0088] Mg 2+ (50mmol·L -1 MgSO 4 ) with 7 different treatments at final concentrations of 0.25 μL, 0.5 μL, 1.0 μL, 1.5 μL, 2.0 μL, 2.5 μL, and 3.0 μL, and the amplification curves are arranged from top to bottom according to the fluorescence values ​​as follows: Figure 5 As shown, the test results show that the earliest peak is 1.5μL, so it is determined that MgSO 4 The optimal amount to add is 1.5 μL.

...

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Abstract

The invention specifically discloses a real-time fluorescent LAMP detection method and kit for Phytophthora infestans, belonging to the field of molecular biology. According to the invention, proper primers are screened out and a reaction system is optimized, so the real-time fluorescent LAMP detection method for Phytophthora infestans is convenient and fast, has high specificity and high sensitivity, and has the lowest detection limit of 371 pg / [mu]L, which is 10 times of the lowest detection limit of corresponding ordinary PCR; and results can be observed in 40 min. The detection method provided by the invention overcomes a plurality of problems such as complex detection process, high cost and low efficiency in conventional detection of Phytophthora infestans.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular. Real-time fluorescent LAMP detection involving a potato infestans. Background technique [0002] Potatoes are widely cultivated crops in the world, second only to corn, wheat, and rice, and are the fourth largest food crops in the world. Potato late blight (Potato late blight) is a devastating disease caused by Phytophthora infestans Mont.de Bary, which causes the death of potato stems and leaves and tuber rot. huge losses to the industry. The difficulty of preventing and controlling late blight and its impact on potato production have surpassed other crop diseases, and it is regarded as the largest crop disease in the world. Phytophthora infestans (P.infestans Mont.de Bary) can cause latent infection of seed potatoes that cannot be detected by naked eyes. After planting such seed potatoes, it often causes potato rot and dead seedlings, and it can also cause rot during s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/6895C12Q1/04C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 杨志辉朱杰华高雪赵冬梅张岱潘阳杨毅清
Owner HEBEI AGRICULTURAL UNIV.
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