Pestalotia funereal pathogenic bacteria molecule detection kit and use method thereof

A detection kit and molecular detection technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long cycle and achieve the effect of simple and fast operation, strong specificity, and improved efficiency and scope

Inactive Publication Date: 2013-03-06
SICHUAN AGRI UNIV
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AI Technical Summary

Problems solved by technology

Aiming at the long period of biological detection of the pathogen of pine red blight at present, a pair of primers with high specificity were screened out by extracting the total DNA of the needle leaves of pine red blight and designing and screening the ITS base-specific sequence of pine red blight , a sensitive and rapid pathogen molecular kit for plant tissue

Method used

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  • Pestalotia funereal pathogenic bacteria molecule detection kit and use method thereof
  • Pestalotia funereal pathogenic bacteria molecule detection kit and use method thereof
  • Pestalotia funereal pathogenic bacteria molecule detection kit and use method thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: the detection of pine red blight in plant tissue

[0032] (1) After surface disinfection, the seemingly healthy samples were cut into three parts according to the leaf tip, leaf center and leaf base, and transferred to a sterilized mortar respectively, and were extracted by liquid nitrogen grinding method combined with a plant genomic DNA extraction kit. Extraction, the total DNA of the sample is used as a template for the next step of PCR;

[0033] (2) According to the design of the ITS region sequence (AF405299.1) of the bacteria already available in Genbank, a pair of PCR primers (AF-F / R) with high specificity were screened out, respectively:

[0034] Upstream primer F (5'-3'): GTCAACCAGCGGAGGGAT 18bp

[0035] Downstream primer R (5'-3'): CGCCGTTGTATTTCAGGAG 19bp

[0036] (3) 50 μL of PCR reaction system includes: 5 μL pine needle genomic DNA stock solution, 25 μL Premix, 2 μL of each primer (10 μmol / L), ddH 2 O 16 μL. The reaction was carried out o...

Embodiment 2

[0038] Embodiment 2: the specificity amplification of primer to pine wilt

[0039] 50 μL of PCR reaction system includes: 5 μL pine needle genomic DNA stock solution, 25 μL Premix, 2 μL of each primer (10 μmol / L), ddH 2 O 16 μL. The reaction was carried out on an iCycler-PCR instrument, and the program was: denaturation at 95°C for 4 minutes; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, and 35 cycles; extension at 72°C for 7 min, and storage at 10°C.

[0040] Test results

[0041] The specificity of detection: Use primer AF-F / R to carry out PCR reaction simultaneously to pine red blight and the arboreal fungus hyphae DNA that has been reported in the laboratory (reaction results see figure 2 ), the amplified band of about 420bp, while for Pestalotiapsis lespedezae, Pestalotiapsis lawsoniae, Lophodermiμm conigenμm, and loose needles The amplification results of Lophodermi μm pinastri and Fusari μmoxyspor μm showed darker or no band...

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Abstract

The present invention discloses a Pestalotia funereal pathogenic bacteria molecule detection kit and a use method thereof. The kit comprises the following components: 16 muL of ultra pure water d.d.H2O, 2 muL of an upstream primer F (5'-3') (GTCAACCAGCGGAGGGAT), 2 muL of a downstream primer R (5'-3') (CGCCGTTGTATTTCAGGAG), and 25 muL of Premix. With the present invention, rapid Pestalotia funereal detection can be achieved, sensitivity is high, the kit can be used in early plant disease detection, and strong technical supports are provided for development of targeted Pestalotia funereal prevention and control and Pestalotia funereal problem solution in time.

Description

technical field [0001] The invention relates to molecular detection of plant diseases, relates to an early rapid molecular detection method for pine red blight, and in particular to a primer and a detection kit for the molecular detection of pine red blight pathogenic bacteria. Background technique [0002] Pine red blight is the main disease of pine leaves in middle and young forests. It is widely distributed in my country and tends to spread gradually. In addition to damaging Masson pine (Pinus massoniana Lamb), it also infects other pine trees, such as Yunnan pine (Pinus yunnanensis), black pine (P.thunbergii), Huashan pine (Pinus armandii Franch), Huangshan pine (P.huangshanensis), Slash pine (P.elliottii), loblolly pine (P.taeda), coastal pine (P.pinaster), tabulaeformis (P.tabulaeformis), Caribbean pine (P.caribaea), island pine (P.insularis), South Asia pine (P.latteri), larch (Larix sp.), money pine (Pseudolarix amabilis). Among the various hosts, Masson pine, Yunn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 刘应高潘欣张岩袁川王欢僬天敏朱天辉
Owner SICHUAN AGRI UNIV
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