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Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for pear fire blight pathogenic bacteria

A technology for detection of Phytophthora amylovora and detection primers, which is applied in the directions of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problem of inability to detect all strains of bacteria

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

AI Technical Summary

Problems solved by technology

When using ELISA to detect this kind of bacteria, the detection limit is about 5×10 3 cfu / Ml, but it is worth noting that the monoclonal antibody method cannot detect all strains of the bacteria

Method used

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  • Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for pear fire blight pathogenic bacteria
  • Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for pear fire blight pathogenic bacteria
  • Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for pear fire blight pathogenic bacteria

Examples

Experimental program
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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 bacterial strain of E. amylovora by reporting fluorescent signals through fluorescent chimeric PCR. During the amplification process, a fluorescent PCR instrument is used to measure the real-time fluorescent light intensity, and the synthetic DNA fragment is measured after the PCR reaction. Melting temperature, transfer the data to the computer for analysis through the supporting software of the fluorescent PCR instrument, the fluorescence generated by the specific amplification of E. amylovora can be observed, and the specific fragments generated by the amplification of E. amylovora can be obtained. The held melting curve (main peak 84 ± 0.5). The results show that the primers and the kit of the invention can accurately detect the positive samples carrying E. amylovora without false negatives.

Embodiment 2

[0036] Example 2: Negative Control

[0037] With the primers and kit of the present invention, according to the detection steps of the present invention, the bacterial wilt of corn (Erwinia stewartii), soft rot of cabbage (Erwinia carotovora subsp.carotovora), bacterial wilt of cassava (Xanthomonas axonopodis pv.manihotis) , Clavibacter michiganensis subsp.michiganensis, and Pseudomonas syringae pv.pisi and other five negative control strains were tested for fluorescence, and a fluorescent PCR instrument was used for real-time fluorescence light intensity measurement during the amplification process , and measure the melting temperature of the synthesized DNA fragments after the PCR reaction finishes, the data is transmitted to the computer and analyzed by the supporting software of the fluorescent PCR instrument, and no similar melting curve (main peak 84 ± 0.5), and the fluorescent signal of the positive control reporter plasmid and the resulting melting curve can be observe...

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Abstract

The invention relates to a real-time fluorescent PCR (polymerase chain reaction) quick detection primer and kit for pear fire blight pathogenic bacteria. 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 an ITS gene sequence of pear fire blight pathogenic bacteria, and a specific amplification primer thereof is designed. The conserved gene sequence is shared by different pear fire blight pathogenic bacterium strains to ensure the reliability in detection of pear fire blight pathogenic bacteria 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 E. amylovora and a kit containing the primer. Background technique [0002] Erwinia amylovora was first discovered in an orchard near New York State in the United States in 1780. Later, it moved westward with American immigrants and arrived in California in 1902. It was discovered in Canada in 1904, entered New Zealand in 1919, and entered New Zealand in 1957. It was introduced to the UK, took root in continental Europe in the 1960s and was introduced to Egypt, and then to Asia in the 1980s. Fire blight caused by this fungus has caused major losses worldwide. Such as U.S. California, the occurrence of pear trees due to the disease in one year is reduced from 125,000 to 1,500. In Joachimstal in its south, about 94% of the pear trees were lost in 4 years, so that they had to give up planting pear trees. [0003] E. amylovora is...

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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