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Fluorescent quantitative PCR (polymerase chain reaction) detection reagent and kit for Claviceps purpurea and their application

A technology of fluorescence quantification and kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of low detection efficiency and no fluorescent signal, and achieve large detection volume and repeatability Good, high sensitivity effect

Active Publication Date: 2019-09-20
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Tm value of ordinary TaqMan probes is 65-72°C, and the fragment length is 25-40 nt. Due to its high specificity, if the probe does not completely match the target gene sequence, the detection efficiency will be low or even no fluorescent signal will occur. problems such as

Method used

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  • Fluorescent quantitative PCR (polymerase chain reaction) detection reagent and kit for Claviceps purpurea and their application
  • Fluorescent quantitative PCR (polymerase chain reaction) detection reagent and kit for Claviceps purpurea and their application
  • Fluorescent quantitative PCR (polymerase chain reaction) detection reagent and kit for Claviceps purpurea and their application

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Experimental program
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Embodiment 1

[0063] Embodiment 1, be used for the preparation of the fluorescence quantitative PCR kit that detects ergot pathogen

[0064] 1. Design primers and probes

[0065] The present invention selects the conserved fragment of the translation elongation factor (Elongation factor 1α, EF-1α) region of ergot bacteria as the target, and performs homology analysis and comparison on the ergot bacteria (C.purpurea) and similar species EF-1α sequences reported in GenBank , select the conserved fragment (59bp) of the ergot EF-1α region, and use primer Express5.0 software to design primers and probes. The synthesis of primers and probes adopts the chemical synthesis method of β-acetonitrile phosphorous acid amine, and the synthetic probe of OligoDNA is carried out by using an automatic DNA synthesizer, and the two ends of the synthesis are fluorescently labeled at the same time. The fluorescent reporter group labeled at the 5′ end of the probe is FAM, labeled at the 3' end with the non-fluor...

Embodiment 2

[0074] Embodiment 2, fluorescence quantitative PCR detects ergot bacteria

[0075] 1. Detection method and result judgment standard

[0076] 1. C.purpurea fluorescence quantitative PCR amplification

[0077] C.purpurea fluorescent quantitative PCR uses a 10 μL volume reaction system, in the ABI7900 fluorescent quantitative PCR instrument, the reaction system is: 5 μL Universal PCR Master Mix, 0.2 μL CP-F (10 μmol / L), 0.2 μL CP-R (10 μmol / L), 0.7 μL CP-P (10 μmol / L), 1 μL genomic DNA, make up to 10 μL with sterilized deionized water . An equal volume of water was used instead of genomic DNA as a blank control. At the same time, set 2 tubes of positive control and negative control 10 μL reaction system.

[0078] The PCR reaction was carried out according to the following reaction parameters: 50°C preheating for 2 minutes; 95°C pre-denaturation for 10 minutes; 95°C for 15 seconds, 60°C for 1 minute, 40 cycles.

[0079] Using the positive ergot bacteria genomic DNA, accordin...

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Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) detection reagent and kit for Claviceps purpurea and their application. A complete single-stranded DNA for detecting Claviceps purpurea is provided herein and is composed of a primer pair and a single-stranded DNA probe; the primer pair is composed of a single-stranded DNA shown as SEQ ID No. 1 and a single-stranded DNA shown as SEQ ID No. 2; the single-stranded DNA probe has a nucleotide sequence of SEQ ID No. 3. The primer pair and the probe having high amplification efficiency and good specificity are constructed and screened through the combination of PCR technique and fluorescent detection; the defect that conventional PCR has high time consumption, high proneness to causing contamination, requirement for electrophoretic detection after amplification and the like is overcome; the reagent and kit of the invention are suitable for qualitatively and quantitatively detecting Claviceps purpurea in a sample quickly and accurately, and have the advantages of good simplicity, good operational convenience, good visibility of results, high sensitivity, good repeatability and the like.

Description

technical field [0001] The invention relates to the detection field of pathogenic fungi, in particular to a fluorescent quantitative PCR detection reagent for ergot pathogens, a detection kit and an application thereof. Background technique [0002] Claviceps purpurea belongs to the phylum Fungi, the subphylum Ascomycota, the class Sclerotinia, the order Spheroides, the family Ergotaceae, and the genus Ergot. It parasitizes in the ovary of gramineous plants such as rye, wheat, barley, and oats, and turns the ovary into sclerotia. Because it is shaped like a grain of wheat, it is called ergot. Ergot fungus infects many cereals and grasses. [0003] There are two aspects to the harm of ergot. On the one hand, ergot disease caused by ergot bacteria is an important disease of wheat, which causes a substantial reduction in crop yield. The yield loss of rye is generally 5%, and the yield loss of wheat is generally 10%. Economic losses also include unusable or downgraded contamin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11C12Q1/6851C12R1/645
CPCC12Q1/6895C12Q1/6851C12Q2563/107C12Q2561/113C12Q2531/113
Inventor 段维军李雪莲张慧丽段丽君郭立新陈先锋
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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