Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for Xanthomonas campestris pv. campestris

A rapid technology for Xanthomonas rapae, applied in the direction of fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as application limitations, false negatives, and obstacles to the research of the final limit of ELISA detection

Inactive Publication Date: 2013-05-22
郑媛
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the process of detection, ELISA detection showed serious limitations: the results of some susceptible stem tissues were false negative, some cassava bacterial wilt strains did not undergo enzyme-linked reactions, and some Xanthomonas pathogenic Mutant pathogens such as citri, euphorbiae, and vasculorum cross-react
These limitations not only hinder the study of the ultimate limit of ELISA detection, but also greatly limit its application

Method used

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  • Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for Xanthomonas campestris pv. campestris
  • Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for Xanthomonas campestris pv. campestris
  • Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for Xanthomonas campestris pv. campestris

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Effect test

Embodiment 1

[0034] Embodiment 1: Effect detection of the present invention

[0035] The specific primers of the present invention detect the standard strain of xanthomonas cassava pathogenic variants of xanthomonas rapeseed by reporting fluorescent signals through fluorescent chimeric PCR, use a fluorescent PCR instrument to measure the real-time fluorescent light intensity during the amplification process, and measure it after the PCR reaction ends The melting temperature of the synthesized DNA fragments is transmitted to the computer and analyzed by the supporting software of the fluorescent PCR instrument, and the fluorescence generated by the specific amplification of the Xanthomonas oleracea cassava pathogenic variety can be observed, and the detection of the Xanthomonas oleracea cassava pathogenic species can be observed, and the The melting curve (main peak 86°C ± 0.5°C) held by the specific fragment produced by the amplification of pv. cassava pv. The results show that the primers...

Embodiment 2

[0036] Example 2: Negative Control

[0037] With primer of the present invention and test kit, by detection step of the present invention, to Xanthomonas campestris pv. Malvacearum pathogenic variety (Xanthomonas campestris pv. pv.holcicola), Xanthomonas campestris pv.holcicola, Xanthomonas oryzae, Xanthomonas badrii, Xanthomonas campestris pv.holcicola Xanthomonas campestris, Xanthomonas campestris pv. amaranthicola, Pseudomonas syringae pv. pisi, Erwinia amylovora Negative control strains are tested for fluorescence. During the amplification process, a fluorescent PCR instrument is used for real-time fluorescence light intensity measurement, and the melting temperature of the synthesized DNA fragments is measured after the PCR reaction is completed, and the data is transmitted to the computer for analysis through the supporting software of the fluorescent PCR instrument. , no melting curve (main peak 86°C ± 0.5°C) similar to that obtained by the amplification of xanthomonas...

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Abstract

The invention relates to a real-time fluorescent PCR (polymerase chain reaction) quick detection primer and kit for Xanthomonas campestris pv. campestris. The primer comprises a forward primer of which the sequence is SEQ ID NO:1 and a reverse primer of which the sequence is SEQ ID NO:2. In the invention, a specific conserved sequence is obtained through molecular biological analysis according to a pthBXam and Tn3 gene sequence of Xanthomonas campestris pv. manihotis, and a specific amplification primer thereof is designed. The conserved gene sequence is shared by different Xanthomonas campestris pv. manihotis strains to ensure the reliability in detection of Xanthomonas campestris pv. manihotis from different sources on a species level. Besides, the primer provided by the invention adopts fluorescence labeling; and compared with the common PCR technology, observation does not need to be performed through a gel electrophoresis method, thus realizing the integrated sealed detection in the detection process.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fluorescent PCR rapid detection primer for Xanthomonas rape pathogenic species and a kit containing the primer. Background technique [0002] Xanthomonas rapeusae pv. cassava is a pathogenic variety of Xanthomonas oleracea. It is the most serious infection disease of cassava crops. Cassava production has caused irreparable losses many times, and with the introduction of foreign high-quality varieties, along with the colonization and breeding promotion of these propagation materials, it has spread and invaded my country. And gradually expand the scope of harm, the trend of harm is intensified, and the consequences of harm are becoming more serious. At present, in my country's major cassava producing areas such as Guangxi, Guangdong, and Hainan, driven by the inherent tropical storms in this area, and under the action of environmental factors such as unique terrain, culti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 郑媛
Owner 郑媛
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