LAMP detection primer for sweet potato blast fungus and visual detection method of sweet potato blast fungus

A detection method and a technology for detecting primers, which are applied in the biological field, can solve the problems of long detection cycle, expensive equipment for detection, and poor specificity, and achieve the effects of simple operation, high sensitivity, and reliable results

Active Publication Date: 2018-12-21
漳浦县六鳌全域有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the technical problems to be solved in the present invention is to provide a kind of sweet potato blast fungus for the traditional detection method of sweet potato blast fungus in the prior art, which requires a long detection cycle, poor specificity, and expensive instruments for detection. LAMP detection primers for bacteria, which have the characteristics of rapidity, high sensitivity, and strong specificity, can be used to develop rapid detection kits suitable for on-site detection in field sweet potato production, so as to ensure the healthy production of sweet potatoes

Method used

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  • LAMP detection primer for sweet potato blast fungus and visual detection method of sweet potato blast fungus
  • LAMP detection primer for sweet potato blast fungus and visual detection method of sweet potato blast fungus
  • LAMP detection primer for sweet potato blast fungus and visual detection method of sweet potato blast fungus

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Experimental program
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Effect test

Embodiment 1

[0053] A kind of LAMP visual detection method of sweet potato blast pathogen, described detection method step is as follows:

[0054] Step (1) Design primers according to the specific gene sequence in the genome sequencing results of Ralstonia solanacearum, and use the LAMP primer design software primer explorer 5.0 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design Primer, obtain two pairs of specific primer pairs for 6 regions, described two pairs of specific primer pairs are as follows:

[0055] Outer primer F3: 5'-ACCAGTTAAAGAATGACCCA-3';

[0056] Outer primer B3: 5'-TGGCAATCCAAGGAATCC-3';

[0057] Internal primer FIP: 5'-GTAACGACCATGCCTGTCCAGATTGTTGTCAATGAAATGGGT-3';

[0058] Internal primer BIP: 5'-GCAGGCATTGCAGCATTGTTAACAATGATCACCAAAACGT-3';

[0059] And the above two pairs of specific primers were synthesized by Fuzhou Shangya Biotechnology Co., Ltd.

[0060] The specific gene sequence of the sweet potato blast fungus (Ralstonia solanacearum) is shown in SE...

Embodiment 2

[0076] Specificity verification of LAMP:

[0077] Respectively extract the total DNA of positive samples infected with sweet potato tuber rot fungus, tomato solanacearum, eggplant solanacearum, capsicum solanacearum, potato solanacearum, and tobacco Ralstonia solanacearum as templates. The extraction method is the same as in Example 1. The 4 pairs of primers designed in Example 1 were used for LAMP amplification and specific detection.

[0078] Experimental results: In the results of the color reaction, only the reaction product of the sample infected with P. diosminum showed fluorescent green, while the other 5 samples were all orange.

[0079] Take 5 μL of the product and analyze it with 2% agarose gel electrophoresis, and the electrophoresis detection result is as follows: figure 2 , where the lanes are expressed as follows: M: DL2000 DNA marker; 1-6 are in turn positive for infection with sweet potato blast fungus, tomato R. solanacearum, eggplant R. solanacearum, pepper...

Embodiment 3

[0082] LAMP detection verification and sensitivity detection

[0083] In order to investigate the sensitivity of the LAMP detection method of sweet potato blast fungus of the present invention, the applicant adopts 10-fold concentration serial dilution method to dilute the DNA sample in embodiment 1 step (3) to a concentration of 10 ng μ L respectively -1 , 1ng·μL -1 , 100pg·μL -1 , 10pg·μL -1 , 1pg·μL -1 , 100fg·μL -1 , 10fg·μL -1 ; 1 fg μL -1 A total of 8 different concentration gradients. Then LAMP and PCR methods were used to detect, and then display reaction and 2% agarose gel electrophoresis.

[0084] Experimental results: It was observed in the color reaction that the concentration of the DNA sample was 10ng·μL -1 , 1ng·μL -1 , 100pg·μL -1 , 10pg·μL -1 , 1pg·μL -1 , 100fg·μL -1 , LAMP amplification products all showed green fluorescence, which was judged as positive;

[0085] For electrophoresis results, refer to Figure 3-4 , image 3 The sensitivity el...

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Abstract

The invention provides a LAMP detection primer for sweet potato blast fungus and a visual detection method of the blast fungus, wherein a group of LAMP primers F3, B3, FIP and BIP of sweet potato blast fungus are mainly designed; LAMP is used for constant temperature amplification; after the amplification reaction, chromogenic reaction or agarose gel electrophoresis detection can be performed to directly and visually observe characteristics of ladder patterns in fluorescent green or electrophoresis. Loop-mediated isothermal amplification technology established by the invention for the sweet potato blast fungus can realize rapid detection of the sweet potato blast fungus in diseased tissues (plants and potato pieces), has advantages of rapid amplification, high efficiency, good specificityand convenient operation, no need of special instruments, and provides a reliable technical basis for prevention and control of the sweet potato blast fungus.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP detection primer for Pyrella diosminum and a visual detection method thereof. 【Background technique】 [0002] Sweet potato blast caused by Ralstonia solanacearum is a map-borne bacterial disease in sweet potato production, and it is subject to plant quarantine. In recent years, sweet potato blast has been expanding and spreading in the main sweet potato producing areas in southern my country, which seriously threatens the production of sweet potato. The infection process of Pyrophthora diosminum on sweet potato is generally invaded from the wound or natural orifice of the sweet potato root, first colonizes in the cortex, intercellular spaces, etc., and then reproduces in large quantities in the plant vessels and nearby tissues, and secretes extracellular polysaccharides Clogging the ducts and destroying the surrounding tissue eventually causes the plant to wilt ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12Q1/6844C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 邱思鑫李华伟张鸿林志坚刘中华纪荣昌邱永祥李国良林赵淼许泳清罗文彬许国春汤浩
Owner 漳浦县六鳌全域有限公司
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