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Quick Clavibacter michiganensis subsp. michiganensis IC-PCR detection kit, its preparation method and its application method

A technology of IC-PCR and detection kits, applied in biochemical equipment and methods, DNA preparation, bacteria, etc., can solve the problems of long detection cycle, low sensitivity, poor specificity, etc., achieve short detection cycle and improve quarantine level , strong specific effect

Inactive Publication Date: 2012-05-23
上海慧耘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to avoid the deficiencies in the prior art and provide a kind of rapid IC-PCR (Imunocapture-PCR, IC-PCR) detection kit of tomato canker sore, to improve the low sensitivity, poor specificity and detection of existing phytopathogenic bacteria detection methods. Disadvantages of long cycle

Method used

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  • Quick Clavibacter michiganensis subsp. michiganensis IC-PCR detection kit, its preparation method and its application method
  • Quick Clavibacter michiganensis subsp. michiganensis IC-PCR detection kit, its preparation method and its application method
  • Quick Clavibacter michiganensis subsp. michiganensis IC-PCR detection kit, its preparation method and its application method

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Effect test

Embodiment 1

[0038] Embodiment 1: To tomato canker bacterium and non-tomato canker bacterium bacterial strain specificity validation test (see image 3 )

[0039] Collect 1 tomato canker bacterium ( Clavibacter michiganensis subsp. michiganensis , Cmm) and 7 non-tomato canker bacteria strains to verify the specificity of this kit, these 7 strains are Pseudomonas syringae pathogenic type of Pseudomonas syringae ( Pseudomonas syringae pv. syringae , Pss), tomato bacterial leaf spot ( Pseudomonas syringae pv. tomato , Pst), tomato bacterial scab ( Xanthomonas campestris pv. vesicatoria , Xcv), cruciferous vegetable black rot pathogenic variant ( Xanthomonas campestris pv. campestris , Xcc), Soybean bacterium macula macula ( Xanthomonas campestris phaseoli , Xcp ), melon bacterial leaf spot ( Pseudomonas syringae pv. lachrymans , Psl), watermelon bacterial fruit blotch ( Acidovorax avenae subsp. citrulli , Aac). Take 50 μL of pure bacterial solution of each test ...

Embodiment 2

[0041] Embodiment 2: gradient dilution direct PCR detection test of tomato canker bacterium pure bacterial liquid (see Figure 4 )

[0042] After the tomato canker sore standard strain was cultivated for 12 h, its absorbance at 600 nm was measured. A600nm Value=0.25-0.3 (at this time, the concentration of bacterial solution is about 10 8 cfu / mL), take 2 mL of this bacterial solution and dilute it 10 times sequentially into sterile PBS, and mark it as 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 , then take 10 -5 、10 -6 、10 -7 100 μL each of the gradient pure bacterial solution was used for plate colony counting. Take the concentration as 2.4×10 7 -2.4×10 1 Put 5 μL of each cfu / mL gradient pure bacterial solution in a PCR tube, add 31.6 μL deionized water, lyse on a PCR instrument at 99°C for 10 min, then quickly place it on ice for 5 min, add 10×PCR buffer after centrifugation 5μL, 2.5mmol / L dNTPs 4μL, 5U / μL Taq 0.4 μL of DNA polymerase, 2 μL of 5 μmol / ...

Embodiment 3

[0044] Embodiment 3: see Figure 5 , IC-PCR detection test of gradient dilution pure bacterial liquid of tomato canker sore,

[0045] The concentration of tomato canker bacteria was 2.4×10 7 -2.4×10 1 Each 50 μL of cfu / mL gradient diluted pure bacterial solution was placed in the detection PCR tube of this kit, incubated at 37°C for 4 h or overnight at 4°C, then the gradient pure bacterial solution in the PCR tube was discarded, and then washed with PBST for 1- Twice, after discarding the washing solution, drain the remaining liquid in the tube, add 36.6 μL of deionized water to the PCR tube, and then lyse in the PCR instrument at 99°C for 10 min, then quickly place it on ice for 5 min, and centrifuge at high speed for a while after cooling. Then add 10×PCR buffer 5μL, 2.5mmol / L dNTPs 4μL, 5U / μL to the tube Taq DNA polymerase 0.4 μL, 5 μmol / L upstream primer 2 μL, 5 μmol / L downstream primer 2 μL, the amplification conditions are 94°C 2min; 94°C 30s, 62.5°C 45s, 72°C 45s,...

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Abstract

The invention relates to a quick detection kit on the level of clavibacter michiganensis subsp. michiganensis genes, which belongs to the field of plant quarantine and plant protection. The kit is suitable for the field prevention and 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 500 muL of 10*PCRBuffer, 400 muL of 2.5mmol / LdNTPs, 40 muL of 5U / muL TaqDNA polymerase, 200 muL of 5mumol / L upward primer, 200 muL of 5mumol / L forward primer, a tube of deionized water, a tube of PBST, 100 tubes of PCR tube coated by Clavibacter michiganensis subsp. michiganensis specific antibody and a tube of positive control.

Description

technical field [0001] The invention relates to a kit for rapid detection of tomato canker at the gene level, belongs to the field of plant quarantine and plant protection, and is specifically aimed at tomato canker ( Clavibacter michiganensis subsp. michiganensis , Cmm) detection. This kit is suitable for field prevention of tomato canker and rapid detection of tomato canker in 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 become widespread worldwide and has caused huge economic losses. It occurs in Northeast my country, North China, Northwest China and the eastern coastal areas of China. The output of some areas has decreased by more than 80%, resulting in a sharp decline in tomato output in my country. Reduced and caused huge economic losses, and at the same time seriously threatened the healthy development of...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N1/20C12N15/10
Inventor 刘箐闵现华
Owner 上海慧耘生物科技有限公司
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