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Loop-mediated isothermal amplification detection primer for ralstonia solanacearu and detection method for ralstonia solanacearu

A technology for detecting primers and bacterial wilt, which is applied in the fields of identification, prevention and control, and detection of crop diseases, and achieves the effects of good practicability, simple and rapid operation, and high sensitivity.

Inactive Publication Date: 2014-03-05
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, LAMP detection is mainly used in the detection of human and animal pathogens, food safety and environmental sanitation. There are very few reports in the detection of plant pathogens. There are no reports at home and abroad on the application of LAMP method to the detection of peanut R. solanacearum

Method used

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  • Loop-mediated isothermal amplification detection primer for ralstonia solanacearu and detection method for ralstonia solanacearu
  • Loop-mediated isothermal amplification detection primer for ralstonia solanacearu and detection method for ralstonia solanacearu
  • Loop-mediated isothermal amplification detection primer for ralstonia solanacearu and detection method for ralstonia solanacearu

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 The present invention is specific to detection of peanut Ralstonia solanacearum

[0037]1. LAMP-specific detection of Ralstonia solanacearum in peanut

[0038] 1) Using R. solanacearum and 25 other strains as test materials, the DNA of R. solanacearum was extracted by CTAB method. The specific method is as follows: inoculate peanut solanacearum in NA liquid medium, culture overnight at 28°C and 200rpm; take 1.5ml culture and centrifuge at 12000rpm for 2min, discard the supernatant, add 567μl TE buffer to resuspend the bacteria, add 30μl 10% SDS solution and 3μl 20mg / ml proteinase K, mix gently, incubate at 37℃ for 1h; add 100μl 5mol / L NaCl solution and mix well, then add 80μl CTAB / NaCl solution, mix and incubate at 65℃ for 10min Add 800 μl of phenol / chloroform / isoamyl alcohol solution (the volume ratio of the three is 25:24:1) and mix well, centrifuge at 12,000 rpm for 5 minutes, take the supernatant, add 0.6-0.8 times the volume of isopropanol, and mix ...

Embodiment 2

[0045] Example 2 Sensitivity detection of the present invention to peanut Ralstonia solanacearum

[0046] 1. LAMP Sensitivity Detection of Ralstonia solanacearum in Peanut

[0047] The extracted R. solanacearum DNA was diluted to 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1fg and 100 ag with 9 different concentration gradients by using 10-fold concentration serial dilution method.

[0048] 1. Amplify according to the reaction system and conditions in Example 1;

[0049] ② Add 1 μl of chromogenic agent SYBR green Ⅰ to the final amplification product of the LAMP reaction. If green fluorescence is observed in the color development results, it is judged as positive, and orange is judged as negative. Or take 2 μl of the amplification product and use 2% agarose gel electrophoresis to detect it. If the characteristic trapezoidal band of LAMP appears, it is judged as positive, and if no amplification band appears, it is judged as negative.

[0050] 2. Test results:

[005...

Embodiment 3

[0053] Embodiment 3 The present invention is artificially inoculated to the detection of Ralstonia solanacearum plant tissue

[0054] 1. Detection of Plant Tissues Artificially Inoculated with Ralstonia solanacearum

[0055] Ralstonia solanacearum was inoculated by seed soaking method. Two symptomatic plants and three non-symptomatic plants were randomly selected for detection. Genomic DNA of R. solanacearum was used as a positive control, and healthy peanut plants were used as a negative control. The DNA of peanut Ralstonia solanacearum was extracted by NaOH rapid lysis method.

[0056] Perform LAMP detection as follows:

[0057] 1. Amplify according to the reaction system and conditions in Example 1;

[0058] ② Add 1 μl of chromogenic agent SYBR green Ⅰ to the final amplification product of the LAMP reaction. If green fluorescence is observed in the color development results, it is judged as positive, and orange is judged as negative. Or take 2 μl of the amplification ...

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Abstract

The invention discloses a loop-mediated isothermal amplification detection primer for ralstonia solanacearu and a quick detection method for the ralstonia solanacearu, which can be used for detecting the specificity of the ralstonia solanacearu. An LAMP (loop-mediated isothermal amplification) detection primer for the ralstonia solanacearu is designed and comprises outer side primers F3:5'-TCTTGATAAGGCGGGGGT-3' and B3:5'-AAACACCGATCTCTCGATGC-3' and inner side primers FIP:5'-CCAGTTCACGGCAAGATCGCTTTCAAGTCCTACCAGACCCA-3' and BIP:5'-ACCTGCTTTGCAAGCAGGGG-GCGTTTGGCTACCACAAGG-3'. After the ralstonia solanacearu is subjected to isothermal amplification, green fluorescence can be observed or a trapezoid strip of the LAMP characteristic emerges through color development of a developing agent SYBRgreenI or electrophoresis detection of agarose gel. The LAMP primer and the detection method which are disclosed by the invention can be used for quickly, sensitively and accurately detecting the ralstonia solanacearu in plants infected with the ralstonia solanacearu during production practice or detecting the ralstonia solanacearu in the plants in the morbidity latent period and can be also used for performing early diagnosis on field diseases, and monitoring and identifying germs; a reliable technical and theoretical basis can be provided for prevention and treatment of diseases caused by the ralstonia solanacearu.

Description

Technical field [0001] The present invention is the field of crop disease testing, appraisal and prevention technology. LAMP detection primers and rapid detection methods involving a peanuts and procaic bacteria are available.Early diagnosis and monitoring and identification of germs in peanuts. Background technique [0002] Peanuts are blight Ralstonia solandaru A bacterial vascular beam disease.The disease first reported in Indonesia in 1905, and then reported or reported in more than 20 countries and regions.At present, China, Indonesia and Vietnam are more harmful.my country ’s reports on peanuts are started in the late 1930s. It is now mainly distributed in the central and southern regions. 2 The above is the first place in the world.The incidence of peanuts is generally about 10%-30%, and the production is more than 20%; the incidence of severe hometown can be more than 50%, which can even lead to a complete collection.It has been reported in recent years that peanuts have ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/01
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 谢世勇李本金陈庆河游泳丁雪玲刘裴清
Owner INST OF PLANT PROTECTION FAAS
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