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A detection method and reagent for sunflower stem canker on single sunflower seeds

The technology of stem canker and detection reagent is applied in the field of detection of sunflower stem canker on single sunflower seeds, and can solve the problems such as difficult accurate detection and identification of sunflower stem canker, so as to avoid false positives and false negatives, eliminate pollution, reliable results

Active Publication Date: 2020-06-16
INSPECTION & QUARANTINE TECH CENT OF NINGBO ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current detection methods for S. helianthus are limited to morphological detection and PCR detection. Due to technical defects, it is difficult to accurately detect and identify S. helianthus on a single sunflower seed.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1, the extraction of single sunflower seed DNA

[0013] Put a single sunflower seed into a 2.0ml plastic centrifuge tube, and then put a steel ball with a diameter of 7mm into it. After freezing in an ultra-low temperature refrigerator for 10 minutes, use a tissue grinder to crush the sunflower seeds. The frequency is set to 30 times / second, and the time is 1 minute. . After the sunflower seeds were crushed, DNA was extracted with a DNA extraction kit (purchased from Tiangen Company). For specific operations, see the kit instruction manual.

Embodiment 2

[0014] Embodiment 2, the design of the special primer and probe of detection sunflower stem canker pathogen

[0015] The gene sequence of S. helianthus stem canker and its close species β-tubulin was collected by the testing laboratory, combined with the gene sequence of S. helianthus stem canker and its close species β-tubulin from GenBank, and DNAMAN6.0.40 software was used to carry out Comparison analysis, to find out the conserved gene sequence of S. helianthus stem canker, according to the principles of primer and probe design, use the software Primer Express 3.0 to design primers and TaqMan-MGB probes, and then compare the designed primers and probes on NCBI Yes, a pair of primers and a probe are finally determined through screening, and their sequences are:

[0016] Forward primer DHF: GCTCGACACG CGAAAAGG;

[0017] Reverse primer DHR: CCAGAGATGG TTTGCCTGAA G;

[0018] Probe DHP: 5'-FAM-TGGCTCGTAG TCTCG-MGB-3', the 5' end of the probe contains a FAM reporter fluorescen...

Embodiment 3

[0019] Embodiment 3, real-time fluorescent PCR detects the specificity test of sunflower stem canker bacterium

[0020] The specific process includes the following steps:

[0021] 1. Sample source

[0022] Sunflower stem canker pure culture samples, Diaporthe apiculata pure culture samples, Diaporthearecae pure culture samples, Diaporthe biguttusis pure culture samples, Diaporthe ellipicola pure culture samples, Diaporthe fusicola pure culture samples, Diaporthe hongkongensis pure culture samples, Diaporthemahothocarpus pure culture samples;

[0023] 2. Extraction of sample DNA

[0024] Extract DNA from the sample in step 1 through a DNA extraction kit (purchased from Tiangen Company), see the kit instruction manual for specific operations;

[0025] 3. Specific detection

[0026] Using the DNA obtained in step 2 as a template, namely: sunflower stem canker DNA, DiaportheapiculataDNA, Diaporthe arecaeDNA, Diaporthe biguttusisDNA, DiaportheellipicolaDNA, Diaporthe fusicolaDNA...

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Abstract

The invention discloses a detection method for diaporthe helianthi munt.-cvetk.,mihaljc.et m.petrov on a single sunflower seed, and a reagent. A pair of specific primers of the diaporthe helianthi munt.-cvetk.,mihaljc.et m.petrov is adopted for performing target gene segment amplification, and a fluorescent probe capable of being complemented and paired with a template is adopted for performing detection, so that the specificity is improved and false positive and false negative are effectively avoided; and the sunflower seed does not need to be subjected to special treatment during detection, so that DNA on the single sunflower seed can be quickly and efficiently extracted and the demand of detecting the diaporthe helianthi munt.-cvetk.,mihaljc.et m.petrov on the sunflower seed is met. The method has the advantages of high detection speed, low cost, reliable result and large-batch treatment.

Description

technical field [0001] The invention relates to PCR detection of sunflower stem canker, in particular to a detection method and reagent for sunflower stem canker on single sunflower seeds. Background technique [0002] Sunflower stem canker (Diaporthe helianthi Munt.-Cvetk., Mihaljc.et M.Petrov) was first discovered in the Vojvodina region of the former Yugoslavia in the summer of 1980, and then expanded to Europe and the United States. Sunflower stem canker first infects the leaves in the early stage of occurrence, forming brown or brown necrotic spots on the leaf tips or leaf edges, and the lesions can extend to the petioles and stems after extending to the midrib. Light brown spots appear on the sunflower stalks, which rapidly expand into withered yellow to light brown large lesions as the disease progresses. The lesions gradually expand and surround the stems, resulting in the upper part of the stems being dry and easy to break, and the pith of the stems and petioles Ro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/6895C12Q2563/107
Inventor 段维军郭立新段丽君陈先锋
Owner INSPECTION & QUARANTINE TECH CENT OF NINGBO ENTRY EXIT INSPECTION & QUARANTINE BUREAU