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Primer for detecting pseudomonas syringaepv.actinidae through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and detection method

A RT-PCR, kiwifruit technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of undiscovered effect-specific primers, etc., and achieve the effect of high specificity and high detection sensitivity.

Inactive Publication Date: 2018-06-19
INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the detection of kiwifruit canker pathogen Pseudomonas syringaepv.actinidae, no specific primers with good real-time fluorescent RT-PCR detection effect have been found

Method used

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  • Primer for detecting pseudomonas syringaepv.actinidae through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and detection method
  • Primer for detecting pseudomonas syringaepv.actinidae through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and detection method
  • Primer for detecting pseudomonas syringaepv.actinidae through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037](1) The DNA of the extracted kiwifruit canker pathogen Pseudomonas syringae pv.actinidae standard product is carried out 10 times of serial dilution, obtains the DNA of the kiwifruit canker canker pathogen Pseudomonas syringae pv.actinidae of gradient dilution, uses it as a template, shown in SEQ ID NO.1 The DNA molecule shown in SEQ ID NO.2 and the DNA molecule shown in SEQ ID NO.2 are respectively used as forward primer and reverse primer to carry out real-time fluorescent RT-PCR amplification, and the Log value of the template concentration is used as the ordinate, and the Ct value obtained by the amplification is used as The abscissa is used to make a standard curve; the obtained amplification curve is as follows figure 1 As shown, the obtained standard curve is as follows figure 2 As shown, specifically: y=-0.1954x+27.809; R 2 = 0.9104.

[0038] (2) Extract DNA from kiwi fruit, and dilute it to the range of the standard curve gained in step (1); carry out real-ti...

Embodiment 2

[0054] Utilize common PCR method, the primer of embodiment 1 is carried out specificity detection and sensitivity detection, and result is respectively as follows image 3 and Figure 4 As shown, it shows that the primers of the present invention have high specificity for the detection of kiwifruit canker pathogen Pseudomonassyringaepv.actinidae. Through BLAST analysis using NCBI gene bank, the E value of the primers of the present invention can be as low as 0.28.

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Abstract

The invention provides a specific primer for detecting pseudomonas syringaepv.actinidae through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The specific primer consists of a forward primer and a reverse primer. The forward primer is: 5'-CCGCTGTGATACAATTCGGC-3', as shown in SEQ ID NO.1, and the reverse primer is: 5'-CGCACATAAGACAACAGGCG-3', as shown in SEQ ID NO.2. The sensitivity of real-time fluorescent RT-PCR detection by the specific primer is at least improved by 100 times compared with that of common PCR detection. The invention further provides a kit based on the specific primer, a detection method and application.

Description

technical field [0001] The invention belongs to the technical field of agriculture and plant quarantine, and specifically relates to a primer for real-time fluorescent RT-PCR detection of kiwifruit canker pathogen Pseudomonas syringaepv. actinidae, a detection method and application thereof. Background technique [0002] Kiwi fruit is known as the king of fruits and the crown of vitamin C. It has high economic benefits. The main domestic planting areas are Sichuan, Shaanxi and Guizhou. The detection, prevention and control of kiwifruit diseases is a necessary means to improve its economic value and promote the economic development of the kiwifruit industry in the region. [0003] Canker is a fatal bacterial disease that seriously threatens the production of kiwifruit, and has been listed as a national forest plant quarantine object. Experts and scholars from the United States, New Zealand, Japan, and China have successively carried out the identification of kiwifruit canker...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/38
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2521/107
Inventor 胡容平石军陈松叶慧丽陈庆东林立金
Owner INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI