Quick clavibacter michiganensis subsp. michiganensis IMS-PCR (Polymerase Chain Reaction) detection kit, preparation method and use method thereof

A detection kit and tomato ulcer technology, applied in biochemical equipment and methods, DNA preparation, bacteria, etc., can solve the problems of long detection cycle, poor specificity, low sensitivity, etc., achieve short detection cycle, strong specificity, The effect of increasing the quarantine level

Inactive Publication Date: 2011-01-19
兰州海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to avoid the deficiencies of the prior art to provide a kind of quick immunomagnetic separation PCR (Immunomagnetic separation-PCR, IMS-PCR) detection kit of tomato canker bacterium, to overcome the low sensitivity of the existing plant pathogen detection method, Disadvantages of poor specificity and long detection cycle

Method used

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  • Quick clavibacter michiganensis subsp. michiganensis IMS-PCR (Polymerase Chain Reaction) detection kit, preparation method and use method thereof
  • Quick clavibacter michiganensis subsp. michiganensis IMS-PCR (Polymerase Chain Reaction) detection kit, preparation method and use method thereof
  • Quick clavibacter michiganensis subsp. michiganensis IMS-PCR (Polymerase Chain Reaction) detection kit, preparation method and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: A rapid IMS-PCR detection kit for tomato canker pathogen, including 1 tube of 10×PCRBuffer, 1 tube of 2.5mM dNTPs, 1 tube of 5U Taq DNA polymerase, and 5μM upstream primer 5′-TCATTGGTCAATTCTGTCTCCC-3′200μL , 5μM downstream primer 5′-TACTGAGATGTTTCACTTCCCC-3′200μL, 1 tube of deionized water, 1 tube of PBST lotion, 1 tube of tomato ulcer pathogen specific immunomagnetic beads, 1 ordinary magnet with a volume of 2mm×3mm×5mm, positive Control 1 tube.

Embodiment 2

[0047] Example 2: See figure 2 , A method of using a rapid IMS-PCR detection kit for tomato canker pathogen, its main features are:

[0048] (1) Enrichment of pathogens: take 1 mL of the test sample and place it in a centrifuge tube, and then add 50 μL of tomato canker pathogen specific immunomagnetic beads into each centrifuge tube, the amount of magnetic beads is 10 5 -10 6 Pieces / mL, shake at room temperature for 2h on a shaker at a speed of 150rpm, then place the test sample on the magnet and let it stand for 5min. When the magnetic beads are collected on the side of the tube wall, carefully suck off the test sample with a pipette. Then wash the centrifuge tube 3 times with PBST for 2 minutes each time, let it stand on the magnet for 5 minutes, and then discard the washing solution;

[0049] (2) PCR amplification: resuspend the immunomagnetic beads enriched with pathogens in 50μL of deionized water, centrifuge and mix well, then transfer to the PCR tube, then put it in the PCR ...

Embodiment 3

[0051] Example 3: A method for preparing specific immunomagnetic beads of a rapid IMS-PCR detection kit for tomato canker pathogen, the main feature of which is that the steps are:

[0052] (1) Dissolve N-hydroxysuccinimide biotin (NHS-Biotin) in dimethylformamide (DMF) and adjust the biotin concentration to 50μg / mL;

[0053] (2) Determine the concentration of the antibody, and dilute the antibody to 10 μg / mL with a carbonate buffer of pH 9.6;

[0054] (3) Mix according to the mass ratio of biotin and antibody at a ratio of 1:10, and shake and react at room temperature for 4 hours;

[0055] (4) Cut the dialysis membrane with a molecular weight of 8000-14000 into small sections with a length of 5-10cm, and then add 500mL 2% NaHCO 3 And 1mmol / L Na with pH 8.0 2 EDTA·2H 2 Boil the dialysis membrane in O for 10 minutes, then thoroughly clean the dialysis membrane with deionized water, and then place it in 1mmol / L Na 2 EDTA·2H 2 Boil it in O for 10 minutes, cool it, and place it in a refrig...

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Abstract

The invention relates to a quick detection kit on the level of clavibacter michiganensis subsp. michiganensis genes, belonging to the field of plant quarantine and plant protection, specially aiming at the detection of clavibacter michiganensis subsp. michiganensis, Cmm. The kit is suitable for the field prevention and the entry and exit plant quarantine of the clavibacter michiganensis subsp. michiganensis as well as the quick detection of the clavibacter michiganensis subsp. michiganensis. The quick clavibacter michiganensis subsp. michiganensis IMS-PCR detection kit is mainly characterized by comprising a 10*PCR Buffer tube, a 2.5mm dNTPs tube, a 5U Taq DNA polymerase tube, 5 mum forward primer 5'-TCATTGGTCAATTCTGTCTCCC-3'200 mul, a 5 mum reverse primer 5'-TACTGAGATGTTTCACTTCCCC-3' 200 mul, a deionized water tube, a PBST washing liquor tube, a clavibacter michiganensis subsp. michiganensis specific immunomagnetic bead tube, an ordinary magnetic block and a positive control tube.

Description

Technical field [0001] The invention relates to a kit for rapid detection at the gene level of tomato canker bacteria, belonging to the field of plant quarantine and plant protection, and specifically for the detection of tomato canker bacteria (Clavibacter michiganensis subsp. michiganensis, Cmm). This kit is suitable for field prevention of tomato canker and rapid detection of tomato canker bacteria in entry and exit plant quarantine and plant protection. Background technique [0002] Bacterial canker of tomato is the most important bacterial disease affecting tomato production. The disease is widely distributed and has been widespread worldwide and has caused huge economic losses. It has occurred in my country's northeast, north, northwest and eastern coastal areas. The output of some areas has decreased by more than 80%, resulting in my country's tomato production The sharp decline caused huge economic losses, and at the same time seriously threatened the healthy development ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N1/20C12N15/10
Inventor 刘箐闵现华
Owner 兰州海关技术中心
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