Fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for plant pathogenic bacterium pantoea ananatis

A detection kit, the technology of Pantoea pineapple, is applied in the field of molecular biology to achieve the effect of a highly specific and accurate detection method

Pending Publication Date: 2021-11-05
上海沃吉基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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There is currently no method for the rapid

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  • Fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for plant pathogenic bacterium pantoea ananatis
  • Fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for plant pathogenic bacterium pantoea ananatis
  • Fluorescent quantitative PCR (polymerase chain reaction) detection method and kit for plant pathogenic bacterium pantoea ananatis

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preparation example Construction

[0026] The preparation process of the positive standard gradient is as follows: take 10 μl of the positive standard plasmid, dilute it in a 10-fold gradient, and sequentially dilute 4-7 gradients to obtain the positive standard gradient.

[0027] After the gradient preparation of the positive standard is completed, the fluorescence quantitative PCR reaction is carried out under the same conditions with the gradient positive standard and sample DNA, and the concentration logarithm of the gradient positive standard is taken as the ordinate, and the Ct value of the experimental result is drawn as the abscissa. , according to the Ct value obtained by the sample test, calculate the concentration of Pantoea pineapple in the sample.

[0028] The present invention also provides a method for detecting the fluorescent quantitative PCR of the plant pathogenic bacterium Pantoea pineapple by the kit, the method comprising: step 1, extracting the genomic DNA of the sample to be tested; step ...

Embodiment 1

[0033] 1. Design of specific primers for detecting Pantoea pineapple.

[0034] Specific primers were designed and synthesized for Pantoea pineapple gyrB gene sequence (GenBank accession number: MW981333.1), and the designed specific primer sequences were analyzed by BLAST. The results showed that the primers had high specificity. Its sequence is as follows:

[0035] Upstream primer: TATCCGCGCCTTTGTTGAGT (SEQ ID NO: 1).

[0036] Downstream primer: ATGCCGTCTTTCTCGGTTGA (SEQ ID NO: 2).

[0037] Design and synthesize positive standard sequences:

[0038] 5- TATCCGCGCCTTTGTTGAGTACCTGAATAAAAACAAAACGCCTATTCACCCAACCGTATTTCTATTTCTCAACCGAGAAAGACGGCAT-3 (SEQ ID NO: 3).

[0039] 2. Extract the genomic DNA of the sample to be tested

[0040] 3. Using the extracted genomic DNA as a template, set a positive standard control and a negative control, prepare a PCR reaction system with the Pantoea pineapple PCR reaction solution and specific primers, and perform a fluorescent quantitative PC...

Embodiment 2

[0049] Example 2. Performance evaluation of fluorescent quantitative PCR detection method.

[0050] 1. Sensitivity assessment of the detection method.

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Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and a kit for plant pathogenic bacteria pantoea ananatis. The kit consists of a PCR reaction enzyme premix solution, a pantoea ananatis specific upstream primer, a downstream primer, a positive control and a negative control. The invention also provides a method for carrying out fluorescent quantitative PCR detection on the plant pathogenic bacterium pantoea ananatis through the kit. The method comprises the following steps of step 1, extracting genome DNA of a sample to be detected; and 2, by taking the genome DNA obtained in the step 1 as a template, setting a positive standard control and a negative control, preparing a PCR reaction system with a Pantoea ananas PCR reaction solution and a specific primer, and carrying out a fluorescent quantitative PCR reaction under optimized reaction conditions. The method has higher specificity, sensitivity and stability when being used for detecting pantoea ananatis in crops and trees, and is a rapid and accurate pantoea ananatis detection method.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection method and a kit in the field of molecular biology, in particular to a fluorescent quantitative PCR detection method and a kit for the plant pathogenic bacterium Pantoea pineapple. Background technique [0002] The plant pathogen Pantoea ananatis is a Gram-negative bacterium, a common epiphytic plant. Pantoea pineapple is distributed all over the world and can be isolated not only from plants but also from the environment. As phytopathogenic fungi, they cause massive economic losses to crops and trees all over the world. It can infect monocotyledonous and dicotyledonous plants, and different hosts can cause different symptoms. For example, infecting pineapple, cantaloupe and other melons can cause fruit rot; infecting corn can cause necrotic spots and stripes; infecting rice can cause rhizome straw rot Wait. Infection of eucalyptus can cause leaf blight and bud tip dieback. Environme...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2545/113C12Q2545/114C12Q2563/107
Inventor 陈美娟陈成成陈冲曹琴琴杨敏陆梦娇王振球章进军贾俊才
Owner 上海沃吉基因科技有限公司
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