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Phomopsis asparagi LAMP (loop-mediated isothermal amplification) detection primer pair and detection method thereof

A technology for stem blight of asparagus and stem blight of asparagus, applied in biochemical equipment and methods, recombinant DNA technology, measurement/inspection of microorganisms, etc., can solve cumbersome detection and identification procedures, high requirements for identification experience, and low accuracy and other problems, to achieve the effect of good practicability, simple and fast operation, and high accuracy

Inactive Publication Date: 2018-02-16
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems in the prior art that the detection and identification procedure of asparagus stem blight is cumbersome, time-consuming, requires high identification experience, low accuracy, and PCR detection needs to rely on amplification equipment and other equipment, a kind of asparagus stem blight is provided. LAMP detection primer set and simple, fast, sensitive and specific visual detection method

Method used

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  • Phomopsis asparagi LAMP (loop-mediated isothermal amplification) detection primer pair and detection method thereof
  • Phomopsis asparagi LAMP (loop-mediated isothermal amplification) detection primer pair and detection method thereof
  • Phomopsis asparagi LAMP (loop-mediated isothermal amplification) detection primer pair and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Design of Asparagus stem blight LAMP primer set

[0031] According to the highly conserved intra-species ribosomal transcribed spacer (rDNA-ITS) sequence of Asparagus stem blight and the variability among families and genus, a LAMP detection primer set with specific amplification effect on Asparagus stem blight was designed. Including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), the nucleotide sequences of which are:

[0032] F3: 5'-ACATTGCGCCCTCTGGTAT-3';

[0033] B3: 5'-CCTACCTGATCCGAGGTCAA-3';

[0034] FIP: 5'-GAAAGCAGTGCCCCAACACCA-CATGCCTGTTCGAGCGTC-3';

[0035] BIP: 5'GGACCCCGAGCGCAGTAGTTA-AGTTGGGGGTTTAACGGC-3'.

Embodiment 2

[0036] Example 2 Establishment of LAMP detection method for asparagus stem blight

[0037] 1. Extraction of DNA from the sample to be tested:

[0038] ① When used to detect pure cultures of pathogenic bacteria, the CTAB method is used to extract the genomic DNA of the tested strains, and the specific steps are as follows:

[0039] (1) Take 0.1 g of mycelium powder in a 1.5 mL centrifuge tube, add 900 μL of 2wt.% CTAB extract, shake and mix with a shaker, put in a water bath at 60°C for 60 min, and centrifuge at 12,000 r / min for 15 min at room temperature;

[0040] (2) Take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (the volume ratio of each is 25:24:1), shake gently, and centrifuge at 8000 r / min for 10 min at room temperature;

[0041] (3) Take 500 μL of the supernatant, add an equal volume of chloroform and extract again, and centrifuge at 8000 r / min for 10 min at room temperature;

[0042] (4) Take 350 μL of supernatan...

Embodiment 3

[0050] Example 3 Asparagus Stem Blight LAMP Detection Specificity Determination

[0051] 1. Using the CTAB method to extract the genomic DNA of 3 strains of asparagus stem blight, asparagus brown spot, asparagus fusarium wilt, soybean anthracnose and tomato cinerea.

[0052] 2. Carry out LAMP amplification using the DNA extracted from the tested bacteria as a template: LAMP reaction system 25 μL, the reaction system includes 0.2 mmol / L of F3 and B3, 1.6 mmol / L of FIP and BIP, and 8 U of Bst DNA polymerase , DNA template 50~100 ng, 12.5 uL LAMP reaction mixture (40 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 1.6 mol / L betaine, 2.0 mM dNTPs, 0.2% Trion X-100), make up 25 uL with sterile ultrapure water. The LAMP reaction conditions were incubation at 63-65°C for 45-60 min and inactivation at 85°C for 5-10 min.

[0053] 3. Determination of LAMP reaction results: Visual observation with fluorescent dyes or agarose gel electrophoresis. The fluorescent dye vi...

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Abstract

The invention provides a phomopsis asparagi LAMP (loop-mediated isothermal amplification) detection primer pair and a detection method thereof, which are specially used for specific detection of phomopsis asparagi, and belongs to the field of disease detection and biological technologies for crops. The phomopsis asparagi LAMP detection primer pair comprises outer primers F3 and B3 and inner primers FIP and BIP and has the sequence in SEQ ID NO.1-4. According to the phomopsis asparagi detection method established on the basis of the primer pair, green fluorescence or ladder bands with the LAMPfeatures can be observed through a chromogenic reaction or agarose gel electrophoresis detection after LAMP constant-temperature amplification. According to the LAMP detection primer pair and the detection method thereof, rapid, sensitive and accurate detection of plants infected with phomopsis asparagi can be realized in production practice, besides, the LAMP detection primer pair and the detection method can be used for early diagnosis of field diseases and monitoring and identification of germs and reliable technological and theoretical bases are provided for early warning and control of the phomopsis asparagi.

Description

technical field [0001] The invention relates to a LAMP detection primer set of asparagus stem blight and a detection method thereof, which is specially used for rapid molecular detection of asparagus stem blight, and can realize early diagnosis of asparagus stem blight and monitoring and identification of pathogens, and belongs to crop diseases Detection, identification, control and biotechnology fields. Background technique [0002] Asparagus is rich in saponins, sterols, flavonoid glycosides, isoflavones, sulfur-containing amino acids, histones, nucleic acid and enzyme macromolecules, vitamin C, vitamin E, B vitamins, rutin, folic acid, carbohydrate immune activators and Manganese, zinc, copper, iron and other trace minerals necessary for human health have extremely high nutritional value and are a kind of precious health-care vegetables. The nutritious and healthy high-end vegetables that are loved by consumers enjoy the reputation of "King of Vegetables" internationally, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/6848C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 石妞妞杜宜新阮宏椿陈福如甘林杨秀娟代玉立
Owner INST OF PLANT PROTECTION FAAS
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