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Primer for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on soft rot pathogenic bacteria of kiwi fruits and method of primer

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

Inactive Publication Date: 2018-06-19
MIANYANG TEACHERS COLLEGE +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 soft rot pathogen Botryosphaeria dothidea, no specific primers for real-time fluorescent quantitative PCR detection with better effect have been found.

Method used

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  • Primer for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on soft rot pathogenic bacteria of kiwi fruits and method of primer
  • Primer for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on soft rot pathogenic bacteria of kiwi fruits and method of primer
  • Primer for RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on soft rot pathogenic bacteria of kiwi fruits and method of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) The DNA of the extracted kiwifruit soft rot pathogen Botryosphaeria dothidea standard substance is carried out 10 times of gradient dilution, obtains the DNA of the kiwifruit soft rot pathogen Botryosphaeria dothidea of ​​gradient dilution, with it as template, with the DNA shown in SEQ ID NO.1 Molecule and the DNA molecule shown in SEQ ID NO.2 are used as forward primer and reverse primer respectively for real-time fluorescent quantitative PCR amplification. 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, Make a standard curve; the obtained amplification curve is as follows figure 1 As shown, the obtained standard curve is as follows figure 2 Shown, specifically: y=-0.050x+29.28, R 2 = 0.482.

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

Embodiment 2

[0054] Utilize common PCR method, the primer of embodiment 1 is carried out specific detection, the result is as follows image 3 As shown, the primers of the present invention have high specificity for the detection of kiwifruit soft rot pathogen Botryosphaeria dothidea.

[0055] Through the BLAST analysis of the 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 real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection on soft rot pathogenic bacteria of kiwi fruits. The primer consists of a forward primer and a reverse primer, wherein the forward primer has a sequence of 5'-TTCAGCCTCGGATTACGGTC-3' as shown in SEQ ID NO.1; the reverse primer has a sequence of 5'-CTCGTGCATGGTGGTCTTGT-3' as shown in SEQ ID NO.2. By adopting the specific primer, the sensitivity of real-time fluorescent RT-PCR detection is at least improved by 100 times when being compared with that of PCR detection. The invention further provides a kit, a detection method and application of the specific primer.

Description

technical field [0001] The invention belongs to the technical field of agriculture and plant quarantine, and in particular relates to a primer for real-time fluorescent RT-PCR detection of kiwifruit soft rot pathogen Botryosphaeria dothidea, 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] Soft rot is one of the main pathogens of kiwifruit diseases. The pathogen has high spore-producing ability from mid-June to the end of September. When kiwifruit young fruits are infected with this fungus, it will seriously affect the growth of the fruit. When infected in June, pre-harvest fruit drop is pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2561/113C12Q2563/107C12Q2521/107C12Q2545/114
Inventor 李艳石军蒲博
Owner MIANYANG TEACHERS COLLEGE