Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A technology of RT-LAMP, building orchid leaves, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc. Application prospects, high application value, rapid and efficient amplification
Inactive Publication Date: 2016-12-07
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
View PDF4 Cites 1 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
Problems solved by technology
The traditional electron microscopy method is time-consuming and laborious, and requires experienced professionals to identify it. ELISA is currently a more commonly used method, but it also has the defect of low sensitivity. PCR
Method used
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0032] To build an orchid mosaic virus RT-LAMP detection method, the steps are as follows:
[0033] Step 100: According to the relatively conservative CP gene sequence of CyMV published by GenBank, use the LAMP primer design software primer explorer 4.0 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design primers for the target gene, and obtain 6 targets 4 specific primers for the region. The 4 specific primer pairs are specifically:
[0034] Outer primer F3: 5'-TGGTTTCCAGGAGGATACCC-3';
[0035] Outer primer B3: 5'-AAGATGGCCCTTGGTGACCT-3';
[0036] Internal primer FIP: 5'-ATTCAGCAGGCTCCAGCGCGGCA-gaattc-CCGCCTTTGACTTCTTCGATGC-3';
[0037] Internal primer BIP: 5'-AAGTACGGCGCCCTTGCCCGT-gaattc-GTGATGAGGTTGCCGTTTTGGA-3'.
[0038] The CP gene sequence of the orchid mosaic virus is shown in SEQ ID NO.1; the nucleotide sequence of the outer primer F3 is shown in SEQ ID NO.2, and the nucleotide sequence of the outer primer B3 is shown in SEQ ID NO.2 ID NO.3; the nucleotide s...
Embodiment 2
[0048] In order to confirm the correct amplification of the LAMP reaction, EcoRI restriction sites were introduced into FIP and BIP when designing LAMP primers in this experiment. After the LAMP reaction product in step 400 of Example 1 was digested with EcoRI enzyme at 37°C overnight, the digested product was run on the gel, and the electrophoresis results were as follows: figure 2 As shown, each lane is indicated as follows: M: DL2000 DNA marker, 1: restriction product, 2: negative control, 3: LAMP product. figure 2 It indicated that the ladder-like band amplified by LAMP could be successfully digested by EcoRI, and the size of the digested product was similar to the expected size.
[0049] In order to further verify the correctness of the LAMP product, the digested product was gel recovered, connected to PMD18-T to construct a recombinant plasmid containing the target fragment, transformed with JOM109 competent cells, and the transformed bacteria were sent for testing.
...
Embodiment 3
[0053] Specificity verification of RT-LAMP:
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
Abstract
The invention provides Cymbidium mosaic virus RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection primers and a detection method thereof. The primers are as follows: the nucleotide sequence of the outer primer F3 is disclosed as SEQ ID NO.2, the nucleotide sequence of the outer primer B3 is disclosed as SEQ ID NO.3, the nucleotide sequence of the inner primer FIP is disclosed as SEQ ID NO.4, and the nucleotide sequence of the inner primer BIP is disclosed as SEQ ID NO.5. The method comprises the following steps: designing primers by using CP gene sequence of Cymbidium mosaic virus as the target gene to obtain 4 specific primers for 6 regions, replacing the polymerase with a DNA chain, carrying out RT-LAMP on the target gene under homothermal conditions by using the specific primers, and finally, carrying out 1% agarose gel electrophoresis analysis, DNA fluorescent dye detection, enzyme digestion verification and clone sequencing comparison on the reaction product. The LAMP technique established according to the Cymbidium mosaic virus has the advantages of high amplification speed and efficiency, favorable specificity, no need of special apparatuses and the like, is simple to operate, has higher application value, and thus, has favorable application prospects in base layer and field detection.
Description
【Technical field】 [0001] The invention relates to a plant virus detection method, in particular to a Jianlan mosaic virus RT-LAMP detection primer and a detection method thereof. 【Background technique】 [0002] Cymbidium mosaic virus (CyMV) is one of the most serious and common viruses that harm orchids. Cymbidium mosaic virus (Cymbidium, Dendrobium, Oncidium, etc.) mainly infects Cymv alone. Orchid mosaic virus infects orchids, which will affect the ornamental and utilization value of orchids, causing symptoms such as mosaic leaves, deformities, petal discoloration, and short flower spikes, resulting in a serious decline in quality, thereby affecting the development of the orchid industry. At present, there is still a lack of effective agents for the prevention and treatment of Jianlan mosaic virus. Cultivation of non-toxic seedlings or targeted isolation and destruction of diseased plants are important measures for the prevention and treatment of viral diseases. This requ...
Claims
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more