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LAMP detection primer group, kit and method for detecting bacterial canker of tomato based on cytC genes

A detection kit, a technology for tomato ulcers, applied in the field of molecular biology, can solve the problem of not designing loop primers and the like, and achieve the effects of simple operation, high sensitivity and strong specificity

Inactive Publication Date: 2016-01-06
吉林省蔬菜花卉科学研究院
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Problems solved by technology

[0005] Although there are reports on detection methods and kits for the detection of tomato bacterial canker by using LAMP technology, most of the detection primers used are designed according to the ITS sequence of tomato canker sores, and no loop primers are designed.

Method used

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  • LAMP detection primer group, kit and method for detecting bacterial canker of tomato based on cytC genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 kit and detection method thereof.

[0035] Prepare tomato canker sores according to the following formula cyt C gene LAMP detection kit, the specification of each kit is 100 reactions:

[0036] (1) Detection primer solution: Synthesize outer primer 1, outer primer 2, inner primer 1, inner primer 2, and loop primer, and prepare dry powder of primers with sterilized deionized water or ultrapure water to prepare mother solutions with a concentration of 100 μmol / L , then take 5 μL of outer primer 1, 5 μL of outer primer 2, 40 μL of inner primer 1, 40 μL of inner primer 2, and 5 μL of loop primer, mix well, and prepare 100 μL of LAMP detection primer solution, wherein the primer sequences are:

[0037] Outer primer 1: TATGCCGCCGGTGACG (SEQ ID NO: 1);

[0038] Outer primer 2: CCATCCGTGTGCCTGCA (SEQ ID NO: 2);

[0039] Internal primer 1: ATCGACGGCGTTGGTCCGG-GGTGGAATCCGCTCTTCAC (SEQ ID NO: 3);

[0040] Internal primer 2: ATCCACGTAGCCGAACGCATCG-GCCCGAACTGGTCAGA...

Embodiment 2

[0051] Embodiment 2 The specificity experiment of kit and detection method.

[0052] Experiments were carried out on the specificity of the kit described in Example 1 and its detection method, and the test samples included: tomato canker, Pseudomonas solanacearum, Pseudomonas solanacearum, potato ring rot, cucumber horn spot fungus, watermelon fruit spot fungus, potato scab fungus.

[0053] In this example, only the sample reaction tube containing P. tomato canker was green, indicating that the kit and detection method of the present invention have good specificity for P. tomato.

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Abstract

The invention discloses a LAMP detection primer group, kit and method for cytC genes in bacterial canker of tomato. The detection primer group consists of five specific primers with the nucleotide sequences respectively represented by SEQ ID NO: 1-5. The detection kit comprises a detection primer solution, DNA polymerase with chain replacement activity, a 10*reaction buffer solution, a dNTPs solution and a color developing agent. The detection method comprises the steps of amplifying a DNA template of a sample at 63-65 DEG C by virtue of the specific primers and the DNA polymerase with the chain replacement activity, and judging whether the sample contains the bacterial canker of tomato by observing the color change of the color developing agent. Special instruments are not required, and the LAMP detection primer group, the detection kit and the detection method have the characteristics of rapidness, high efficiency, simplicity and convenience in operation, strong specificity, high sensitivity and the like and are applicable to field detection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a detection method for plant diseases, in particular to a primer set, a kit and a detection method for rapidly detecting tomato canker bacterium by using a loop-mediated isothermal amplification technique (LAMP). Background technique [0002] Tomato bacterial canker (Bacterial cankeroftomato) is caused by Corynebacterium michigani subspecies ( Clavibacter michiganensis subsp. Michiganensis ) is a vascular disease caused by seeds and soil. Since it was first discovered on greenhouse tomatoes in Michigan, USA in 1909, it has been widely distributed in more than 60 countries and regions around the world, and it has occurred seriously in Beijing, Heilongjiang, Jilin, Liaoning, Inner Mongolia and other places in my country. It is on the rise and becomes a devastating disease in tomato production, causing yield losses of up to 80%. In 1995, my country included the pathogen in t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107
Inventor 毛芙蓉王利波刘燕妮王学国许世霖郑士金郑建超
Owner 吉林省蔬菜花卉科学研究院
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