Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

IC-RT-LAMP (loop-mediated isothermal amplification) detection method for specially detecting LNYV (Lettuce necrotic yellows virus)

An IC-RT-LAMP, detection method technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of complex operation, expensive equipment and high technical level requirements, and achieve simplified operation process, The effect of reducing detection difficulty and improving detection efficiency

Pending Publication Date: 2019-04-26
NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Purpose of the invention: In view of the problems in the prior art that the detection method of lettuce necrosis and yellowing virus LNYV is complex in operation, requires high technical level of operators, and the required equipment is expensive, the purpose of the present invention is to provide a detection method for lettuce necrosis and yellowing virus LNYV. IC-RT-LAMP specific detection primer composition; Another object of the present invention is to provide the IC-RT-LAMP detection kit of above-mentioned lettuce necrotic yellowing virus LNYV; Another object of the present invention is to provide above-mentioned lettuce necrotic yellowing IC-RT-LAMP detection method of virus LNYV

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
  • IC-RT-LAMP (loop-mediated isothermal amplification) detection method for specially detecting LNYV (Lettuce necrotic yellows virus)
  • IC-RT-LAMP (loop-mediated isothermal amplification) detection method for specially detecting LNYV (Lettuce necrotic yellows virus)
  • IC-RT-LAMP (loop-mediated isothermal amplification) detection method for specially detecting LNYV (Lettuce necrotic yellows virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: the acquisition of positive control substance

[0064] 1. Extraction of total RNA

[0065] The lettuce leaf that 50-100mg Lanzhou area is infected with LNYV is ground in liquid nitrogen, extracts the total RNA of susceptible tissue with plant total RNA extraction kit;

[0066] 2. Primer design and synthesis

[0067] According to the lettuce LNYV protein N gene sequence (GenBank accession number: AJ746190.1) registered on GenBank, a pair of specific forward (LNYV-F) and reverse primers (LNYV-R) were designed and synthesized; the sequences of the above primers are as follows :

[0068] LNYV-F: 5'-CTAGGGTCAGGAACACAGCG-3'

[0069] LNYV-R: 5'-ATACCATGCCGCGAATCTGT-3';

[0070] 3. Preparation of positive control substance

[0071] 1) RT reaction

[0072] Use lettuce LNYV reverse primer LNYV-R and M-MLV reverse transcriptase to carry out RT reaction to synthesize the first strand of cDNA;

[0073] The 10 μL RT reaction system is as follows: 2 μL total RNA, ...

Embodiment 2

[0083] Example 2: An IC-RT-LAMP detection kit for specific detection of lettuce necrotic yellowing virus LNYV

[0084] The kit consists of LNYV antibody IgG, antibody coating buffer (CB), phosphate buffer (PBS), phosphate wash buffer (PBST), RT reverse primer LNYV-R, LAMP primer set, first-strand cDNA synthesis reagent , LAMP amplification reaction reagent, fluorescent dye detection solution, negative control substance and positive control substance, wherein the specific LAMP primer set includes forward outer primer F3, reverse outer primer B3, forward inner primer FIP, reverse inner primer The sequences of BIP, forward loop primer LF and reverse loop primer LB, the above-mentioned specific RT reverse primer LNYV-R and LAMP primer set are as follows:

[0085] LNYV-R: 5'-ATACCATGCCGCGAATCTGT-3';

[0086] F3: 5'-ACCTAAGCCAGCAATGACAT-3';

[0087] B3: 5'-TGCCCAATCCAGCCTCTT-3';

[0088] FIP: 5'-CTGTTAGATACTCCCGCCTGCGACACCCTCCTAACACCTCCTT-3';

[0089] BIP: 5'-CTCTGGACGCAACCCGGAA...

Embodiment 3

[0096] Example 3: Establishment of method for detection of lettuce necrotic yellowing virus LNYV by immunocapture IC-RT-LAMP

[0097] 1, the preparation method of rabbit anti-LNYV polyclonal antibody IgG among the present invention:

[0098] 1) Expression and purification of LNYV protein N gene fusion protein: total RNA was extracted from lettuce leaves infected with LNYV in Lanzhou area, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the protein N gene fragment of LNYV. Cloned into pET-28a vector by enzyme digestion. The recombinant plasmid was transformed into Escherichia coli BL21, cultured at 37°C, induced by IPTG, and purified by nickel column affinity chromatography to obtain a 22.5kDa LNYV protein N gene fusion protein;

[0099] 2) Preparation of polyclonal antibody IgG: immunize New Zealand white rabbits with 1 mg / mL fusion protein of the above-mentioned LNYV protein N gene as an immunogen;

[0100]In the initial immunization, t...

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

No PUM Login to View More

Abstract

The invention discloses an IC-RT-LAMP (loop-mediated isothermal amplification) detection method for specially detecting LNYV (lettuce necrotic yellows viruses). The method mainly comprises the steps of specially capturing LNYV particles by antibody IgG of LNYV; using specific primers of the LNYV for reverse transcription RT reaction for synthesizing cDNA; then, using specific LAMP primer groups toperform LAMP amplification; after the reaction is completed, detecting whether a sample to be detected contains LNYV or not by a fluorescent dye naked eye observation method or agarose gel electrophoresis. The method has the advantages that the specificity is high; the sensitivity degree reaches 3.5 pg / mL and is 100 times higher than that of ordinary PCR; the RNA extraction is not needed; the special instrument and equipment is not needed; the operation process is simplified; the detection difficulty is reduced; the detection efficiency is improved; a novel technical platform is provided forthe lettuce LNYV molecular detection; the method can be used for monitoring the occurrence, diffusion and spreading of the LNYV, and is suitable for being popularized and applied to primary places.

Description

technical field [0001] The invention relates to an IC-RT-LAMP kit for specifically detecting lettuce necrotic yellowing virus LNYV and a detection method thereof. Background technique [0002] lettuce( Lactuca sativa ) belongs to the leaf type of lettuce of Compositae, also known as goose dish and lettuce. It belongs to annual and biennial vegetables. In recent years, lettuce has become more and more popular, especially after discovering some of its effects, the planting area of ​​lettuce has gradually expanded. At the same time, growers are also facing a series of disease problems, among which viral diseases are relatively common diseases. widely. Planting in the open field is very harmful, and the incidence rate is as high as 60%, or even higher. [0003] After the virus infects lettuce, it destroys the nutrient channel of lettuce, changes the organs of lettuce or disrupts the balance of tissue metabolism, and inhibits photosynthesis. Plants are more affected by the di...

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
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12R1/94
CPCC12Q1/6804C12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2565/125C12Q2563/131
Inventor 张玉宝王亚军谢忠奎郭志鸿何玉惠邱阳
Owner NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com