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Primer combination for detecting Phialophora gregata by loop-mediated isotherm amplification technique and application thereof

A primer composition and ring-mediated isothermal technology, applied in the biological field, can solve the problems of long cycle, expensive thermal cycle equipment, and long time consumption, and achieve the effects of good practicability, expanded use range, and high accuracy

Inactive Publication Date: 2015-02-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the biological detection method of soybean stem brown rot in the prior art. The time is too long and the operation is cumbersome (the amplification result can only be judged after electrophoresis), and the new molecular detection method and primer composition of soybean stem brown rot provided by LAMP for soybean stem brown rot Detection, short detection period (only 1h), high accuracy, high sensitivity, the detection results can be observed with naked eyes

Method used

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  • Primer combination for detecting Phialophora gregata by loop-mediated isotherm amplification technique and application thereof
  • Primer combination for detecting Phialophora gregata by loop-mediated isotherm amplification technique and application thereof
  • Primer combination for detecting Phialophora gregata by loop-mediated isotherm amplification technique and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Loop-mediated isothermal amplification kit for detecting soybean stem brown rot

[0039] A loop-mediated isothermal amplification kit for detecting soybean stem brown rot, including 1ml detection solution, which uses ultrapure water as a solvent, contains 1.6 μM forward internal primer FIP, 1.6 μM reverse internal primer BIP, 0.2 μM μM forward outer primer F3, 0.2μM reverse outer primer B3, loop primers LB and LF 0.8μM, 1.4mM dNTPs, 0.8M Tris‐HCl (pH 8.8), 0.4mM KCl, 0.4mM (NH 4 ) 2 SO 4 , 0.24mM 4% Triton X‐100, 8mM MgSO 4 , 0.192mM Hydroxybromophenol Blue (HNB), 8U·μL ‐1 Bst DNA polymerase.

Embodiment 2

[0040] Embodiment 2 primer composition specificity investigation

[0041] In order to verify the specificity of the primer composition, that is, the LAMP method established on this basis, select standard soybean stem brown rot bacterial strains, Rhizoctonia solani, soybean purple spot fungus, colloid anthracnose, Fusarium, soybean carbon rot fungus, The DNA of Aspergillus oryzae, Alternaria, flat head anthracnose and Phalaenopsis as template, take 2μL DNA solution, add 25μl kit detection solution, the total volume is 27μl; figure 1 shown. The results show that the specific LAMP primer composition can specifically recognize soybean stem brown rot bacterial strains, and samples containing soybean stem brown rot (tube No. 1 to tube 4) turn sky blue under normal light, while other species (tubes 5 to Tube No. 13) is purple under normal light, and the negative control (No. 14, No. 15) is also purple under normal light.

Embodiment 3

[0042] Example 3 Sensitivity test of soybean stem brown rot fungus LAMP primer

[0043] In order to determine the sensitivity of the LAMP detection method, the extracted DNA of the standard soybean stem brown rot fungus strain was measured with a spectrophotometer (1 μg / μl), then diluted 10 times with DEPC water, and stored at -70°C as a template. Take 2 μl of DNA dilutions of each concentration after 10-fold dilution as a template, add 25 μl of the detection solution of the kit, and the total volume is 27 μl; the reaction procedure is: 64°C for 60 minutes; test under normal light, the result is as follows figure 2 Shown: tubes 1 to 6 turn sky blue under normal light, indicating that the sensitivity of the method of the present invention is 10 pg / μL.

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Abstract

The invention discloses a primer combination for detecting Phialophora gregata by loop-mediated isotherm amplification technique and application thereof. The primer combination is composed of a forward outer primer F3 shown in SEQ ID NO.1, a reverse outer primer B3 shown in SEQ ID NO.2, a forward inner primer FIP shown in SEQ ID NO.3, a reverse inner primer BIP shown in SEQ ID NO.4, a loop primer LF shown in SEQ ID NO.5, and a loop primer LB shown in SEQ ID NO.6. The primer combination can be applied to preparation of a Phialophora gregata loop-mediated isotherm amplification detection kit. The kit has good specificity and sensitivity, has high amplification speed, has high efficiency, can identify conveniently, can meet the present crying need for rapid detection of the Phialophora gregata, can be used for quarantine of imports and exports, field quarantine and other field testing, and can be easily promoted and applied in a large range.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a primer composition for detecting soybean stem brown rot pathogen by a loop-mediated isothermal amplification technique and an application thereof. Background technique [0002] Soybean stem brown rot (Phialophora gregata f.sp.sojae, hereinafter referred to as PGS) infects soybean plants and causes soybean stem brown rot. The disease was first discovered in Illinois, U.S. in 1942, and has been reported many times in North America and Canada soybean-growing areas, causing huge losses in soybean production in the U.S. and other places, with yield losses of up to 30%, up to 66 %. PGS is listed as a type A1 quarantine pest of the European and Mediterranean Plant Protection Organization (EPPO), and it is also one of the plant quarantine harmful fungi in my country. At present, PGS mainly occurs in Brazil, the midwestern and southeastern states of the United States, Canada, Argent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844
Inventor 郑小波吴旭东
Owner NANJING AGRICULTURAL UNIVERSITY
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