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A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting flat head anthracnose

A primer composition and technology of anthrax bacteria, applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms, can solve the problems of few recognition sites for target gene segments, low detection specificity and sensitivity, and cumbersome detection process, etc. Achieve the effects of good practicability, increased detection operation links, and low sensitivity

Active Publication Date: 2017-01-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The known molecular detection target ITS of flat-headed anthrax can not meet the needs of LAMP primer design. The existing molecular detection technology based on common PCR for this pathogen is relatively backward, and it takes a long time to detect the target gene segment. Few, high false detection rate, low detection specificity and sensitivity, cumbersome detection process and other defects

Method used

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  • A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting flat head anthracnose
  • A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting flat head anthracnose
  • A kind of loop-mediated isothermal amplification primer composition and application thereof for detecting flat head anthracnose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 A LAMP detection kit for detecting flat head anthracnose

[0063] Kit reaction system: detection solution and dye SYBR Green I.

[0064] The detection solution preparation method is as follows:

[0065] 0.8 μM forward inner primer Rpb1-FIP, 0.8 μM reverse inner primer Rpb1-BIP, 0.1 μM forward outer primer Rpb1-F3, 0.1 μM reverse outer primer Rpb1-B3, 0.1 μM loop primer Rpb1-LF, 1.4 mM dNTPs , 8mMMgSO 4 , 0.8M Betaine, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4) 2 SO 4 , 4% Triton X-100, 8U·μL -1 Bst DNApolymerase, add ultrapure water to prepare 1mL detection solution, and the shelf life is 1 year.

Embodiment 2

[0066] Embodiment 2 Investigation on the versatility of the kit of the present invention

[0067] Select the standard strain of P. anthracis shown in Table 2, and the DNA of P. anthracis from different regions and different hosts as templates, take 4 μl of DNA solution, add 18 μL of detection solution and 3 μL of sterilized deionized water for LAMP reaction, and the reaction procedure For: 62°C for 70 minutes; add 0.25 μL of dye SYBR Green I as a reaction indicator after amplification, observe the color change of the reaction tube after the reaction, and detect it under normal light. The samples of flat head anthrax bacteria from different hosts and regions all change It is yellow-green, and the negative control is orange under normal light. It shows that this technique is applicable to the detection of flathead anthrax from different hosts and regions. figure 2 Only 8 representative strains were selected and listed.

Embodiment 3

[0069] Select the standard strain of flat-headed anthracnose, its close species G. anthracnose and A. oxysporum, and other pathogenic fungi (Soybean purple spot, Aspergillus oryzae, Alternaria, soybean anthracnose, E. niger, southern stem Canker bacterium, soybean northern stem canker bacterium, soybean stem rot fungus, soybean stem brown rot fungus, soybean Phytophthora bacterium, Fusarium oxysporum, Helminthosporium maize) DNA as a template, get 4 μ l DNA solution, add 18 μL of detection solution and 3 μL of sterilized deionized water were used for LAMP reaction. The reaction program was: 62°C for 70 minutes; after amplification, 0.25 μL of dye SYBR Green I was added as a reaction indicator. After the reaction, the color change of the reaction tube was observed and detected under normal light. Samples containing P. anthracis should turn yellow-green in normal light, while samples containing other species and negative controls should be orange in normal light. The results sho...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof. The primer composition comprises a forward outer primer Rpb1-F3 as shown in SEQ ID NO.2, a reverse outer primer Rpb1-B3 as shown in SEQ ID NO.3, a forward inner primer Rpb1-FIP as shown in SEQ ID NO.4, a reverse inner primer Rpb1-BIP as shown in SEQ ID NO.5 and a loop primer Rpb1-LF as shown in SEQ ID NO.6. By the primer composition, the problems that the biological detection method requires long period, wastes time and energy and is tedious and poor in specificity and a thermal cycling instrument is needed and colletotrichum truncatum cannot be rapidly detected by virtue of the PCR detection technology in the prior art are solved and the primer composition has the advantages of short detection period (only 70 minutes), strong specificity and high sensitivity, and the detection results can be visually observed.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a loop-mediated isothermal amplification primer composition for detecting flathead anthracnose bacteria and its application. Background technique [0002] Soybean anthracnose is an important disease in soybean producing areas in the world. It generally occurs in important soybean producing areas such as Northeast my country, North China, East China, Northwest China, and South China. According to reports, it has occurred in 16 provinces including Heilongjiang, Jilin, and Liaoning. [1] . Soybean anthracnose can occur from seedling stage to harvest stage, and can damage cotyledons, cotyledons, leaves, petioles, stems, pods and seeds. The disease spots on the cotyledons are round, reddish brown to dark brown, and sunken. Lesions on stems are brown or reddish-rust, thin strips, sunken and cracked. The lesions on the leaves of the adult plant stage mainly occur in the veins, which ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q2531/119C12Q2563/107
Inventor 郑小波田擎陆辰晨王帅帅张海峰王源超
Owner NANJING AGRICULTURAL UNIVERSITY
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