Primer pair CM for detecting ciborinia camelliae, detection reagent for detecting ciborinia camelliae and application of primer pair CM for detecting ciborinia camelliae and detection reagent for detecting ciborinia camelliae

A detection reagent and primer pair technology, applied in the field of real-time fluorescent PCR detection, can solve the problems of high professional quality requirements for detection personnel, false negative results, time-consuming and labor-intensive problems, and achieve the goal of avoiding interference from human factors, eliminating pollution, and improving sensitivity Effect

Pending Publication Date: 2020-02-14
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, domestic and foreign methods for detecting camellia rot pathogens are limited to morphological detection methods, which are time-consuming and laborious. It takes more than 15 days to obtain the detection results, and it is easy to miss detection and produce false negative results.
Moreover, in the process of traditional application of morphological detection methods, the professional quality requirements of relevant detection personnel are relatively high.

Method used

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  • Primer pair CM for detecting ciborinia camelliae, detection reagent for detecting ciborinia camelliae and application of primer pair CM for detecting ciborinia camelliae and detection reagent for detecting ciborinia camelliae
  • Primer pair CM for detecting ciborinia camelliae, detection reagent for detecting ciborinia camelliae and application of primer pair CM for detecting ciborinia camelliae and detection reagent for detecting ciborinia camelliae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Preparation of detection reagents for detection of camellia flower rot bacteria

[0055] The detection reagent for detection of camellia rot pathogen provided in this example consists of a primer pair named CM and a probe named CM-P. CM is composed of single-stranded DNA named CM-F and CM-R respectively, and the molar ratio of the two is 1:1. CM can be amplified from the genomic DNA of Rhododendron fusarium wilt to obtain the sequence shown in sequence 4 in the sequence table. DNA fragments. The sequences of each primer are as follows:

[0056] CM-F (forward primer, sequence 1 of the sequence listing):

[0057] Sequence 1: 5'-TCTTGATGGCTCTGGTGTGTAAG-3'.

[0058] CM-R (reverse primer, sequence 2 of the sequence listing):

[0059] Sequence 2: 5'-CAAAACATGTCAGAATCGTTCAAAA-3'.

[0060] The nucleotide sequence of the probe CM-P is shown in Sequence 3 of the sequence listing, with a fluorescent reporter group FAM at the 5' end and a fluorescent quencher group MGB at t...

Embodiment 2

[0065] Test the specificity of the detection reagent prepared in Example 1

[0066] Sample to be tested: Camellia rot pathogen Ciborinia camelliae (strain numbers are CM1, CM2, CM3, CM4, CM5, CM81, CM82, CM83, CM84, CM85, CM96~CM143, CM188, CM189, CM-JX6, CM-JX7, CM -JX8, CM-JX9, CM-JX10, CM-HN6, CM-HN7, CM-HN8, CM-HN9, CM-HN10), Rhododendron wilt Ovulinia azalea (OV70), Botrytis cinerea, Sclerotinia Sclerotinia sclerotiorum, Claviceps purpurea (SC1, SC2, SC3), Monilinia fructicola MF, Sclerotinia laxa ML, Sclerotinia sclerotiorum, Colletotrichum sp. .), Phyllosticta sp., Diaporthe sp., Botryosphaeria sp., Phoma sp. All the above-mentioned strains were isolated and identified in this experiment, and can be purchased from the market, and none of them involved new strains.

[0067] 1. Extract the genomic DNA of the sample to be tested.

[0068] 2. Using the genomic DNA obtained in step 1 as a template, perform real-time fluorescent PCR.

[0069] Reaction system (10μL): 5μL ...

Embodiment 3

[0073] Test the sensitivity of the detection reagent prepared in Example 1

[0074] 1. Extract the genomic DNA of the camellia rot pathogen CM143 strain, and use the genomic DNA as a template after gradient dilution.

[0075] 2. Using the genomic DNA obtained in step 1 as a template, perform real-time fluorescent PCR.

[0076] Reaction system (10μL): 5μL Universal PCR Master Mix, 0.5 μL CM-F (10 μmol / L), 0.5 μL CM-R (10 μmol / L), 0.5 μL CM-P (10 μmol / L), 1 μL genomic DNA, make up to 10 μL. In the reaction system, the concentration of CM-F is 0.5 μmol / L, the concentration of CM-R is 0.5 μmol / L, and the concentration of CM-P is 0.5 μmol / L. An equal volume of water was used instead of genomic DNA as a blank control.

[0077] There are 1-8 reaction system systems in total, and in reaction system 1, the genomic DNA content is 55ng.

[0078] In reaction system 2, the genomic DNA content was 5.5ng.

[0079] In reaction system 3, the genomic DNA content was 550pg.

[0080] In ...

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Abstract

The invention discloses a primer pair CM for detecting ciborinia camelliae, a detection reagent kit for detecting ciborinia camelliae, a detection method for carrying out a real-time fluorescence PCRby means of the primer pair CM for detecting ciborinia camelliae or the detection reagent kit for detecting ciborinia camelliae and relative application of the primer pair CM for detecting ciborinia camelliae and the detection reagent kit for detecting ciborinia camelliae, and particularly belongs to the technical field of biology. The primer pair CM is composed of a primer CM-F and a primer CM-R,wherein CM-F is a single-stranded DNA molecule shown in a sequence 1 of a sequence table as shown in the description or a DNA molecule which is obtained by replacing and / or deleting and / or adding oneor several of amino acids on the sequence 1 as shown in the description and has same functions with the sequence 1 as shown in the description; and CM-R is a single-stranded DNA molecule shown in a sequence 2 of the sequence table as shown in the description or a DNA molecule which is obtained by replacing and / or deleting and / or adding one or several of amino acids on the sequence 2 as shown in the description and has same functions with the sequence 2 as shown in the description. The provided primer pair and kit and a provided probe can detect ciborinia camelliae faster and more accurately.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer pair CM and a detection reagent kit for detecting camellia rot pathogen, a detection method for real-time fluorescent PCR using the same, and related applications. Background technique [0002] The camellia flower rot pathogen belongs to the phylum Fungi, the subphylum Ascomycota, the class Ascomycetes, the order Molliculales, the family Sclerotiniaceae, and the genus Phyllophthora. Camellia rot fungus has been included in the list of imported plant quarantine pests in my country, and it is a quarantine plant pathogenic fungus. At present, camellia rot is mainly distributed in the United States in North America, Germany, Britain, Portugal, Spain, France, Italy, Switzerland and Germany in Europe, New Zealand in Oceania, and Japan in Asia. Camellia rot is an obligate parasite, which only infects the flowers of the genus Camellia and does not infect other parts. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2563/107C12Q2561/113Y02A50/30
Inventor 段维军郭立新吕燕
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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