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Kit used for specific detection of LiLy mottlt virus, and detection method thereof

A technology of lily mottle virus and detection method, which is applied in the direction of microorganism-based method, biochemical equipment and method, microorganism measurement/inspection, etc., can solve the problems of high technical requirements, improve detection efficiency, reduce experiment difficulty, and be easy to operate Effect

Pending Publication Date: 2018-11-13
NORTHWEST INST OF ECO ENVIRONMENT & RESOURCES CAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The technical problem to be solved by the present invention is to overcome the defects and deficiencies in the existing LMoV virus detection method, which require high technical requirements and rely on professional equipment, and provide an IC that can specifically and efficiently detect lily mottle virus without the need for molecular biology professional equipment. -RT-LAMP method; specifically, the combination of immunology and LAMP technology is ingenious. First, the specific antibody of LMoV virus is coated on the wall of the coating container to specifically capture the LMoV particles in the detection sample, which can concentrate and The function of purifying the virus, and then using the captured LMoV particles as a template to perform reverse transcription RT and then LAMP reaction to achieve the purpose of detecting lily mottle virus

Method used

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  • Kit used for specific detection of LiLy mottlt virus, and detection method thereof
  • Kit used for specific detection of LiLy mottlt virus, and detection method thereof
  • Kit used for specific detection of LiLy mottlt virus, and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The acquisition of embodiment 1 positive control substance

[0054] 1. Extraction of total RNA

[0055] Grind 50-100 mg of LMoV-infected leaves of Lily of Lanzhou in liquid nitrogen, and extract the total RNA of the susceptible tissue with a plant total RNA extraction kit.

[0056] 2. Primer design and synthesis

[0057] A pair of specific forward (LMoV-F) and reverse primers (LMoV-R) were designed and synthesized based on the CP gene sequence of Lanzhou lily LMoV (GenBank accession number: MF781080) registered on GenBank; the sequences of the above primers are as follows:

[0058] LMoV-F: 5'-GCTTCCAGTTCCCACACAAG-3'

[0059] LMoV-R: 5'-GTCGTTGAGACCATACTCGT-3'.

[0060] 3. Preparation of positive control substance

[0061] 1) RT reaction

[0062] Using LMoV reverse primers LMoV-R and M-MLV reverse transcriptase from Lily of Lanzhou to carry out RT reaction to synthesize the first strand of cDNA.

[0063] The 10 μL RT reaction system is as follows: total RNA 2 μL, 1...

Embodiment 2

[0073] Example 2 Establishment of the method for detecting lily LMoV by immunocapture IC-RT-LAMP

[0074] 1, the preparation method of rabbit anti-LMoV polyclonal antibody IgG among the present invention:

[0075] 1) Purification of LMoV natural virions: Take 50g of frozen LMoV-infected Lily of Lanzhou leaves, add 200mL 0.5M, pH7.0 potassium phosphate buffer solution containing 0.1% β-mercaptoethanol and 0.01M EDTA, in a frozen mortar Grind until homogeneous, filter with 4 layers of gauze, and take the filtrate; add 200mL chloroform to the filtrate, stir for 15 minutes; centrifuge at 5000g for 15 minutes, keep the supernatant, discard the precipitate; add 7.5% PEG 6000 (W / V) to the supernatant Stir and dissolve with 4% sodium chloride (W / V) and let stand for 30 min; centrifuge at 8000g for 20 min; retain the precipitate, resuspend in 0.5M, pH7.0 containing 0.1% β-mercaptoethanol, 0.01M EDTA and 2% In the potassium phosphate buffer solution of Triton X-100, overnight at 4°C; c...

Embodiment 3

[0097] Example 3 Specificity of Immunocapture IC-RT-LAMP Detection of LMoV

[0098] In order to analyze the specificity of immunocapture IC-RT-LAMP in detecting LMoV, the infected leaves of the three main viruses (lily recessive virus-LSV, cucumber mosaic virus-CMV and LMoV) most commonly infecting lily were taken as samples, respectively. The virions were captured with LMoV polyclonal antibody IgG, and reacted with the IC-RT-LAMP detection system in the above examples. The healthy lily leaves were used as the negative control, and the experiment was repeated 3 times.

[0099] The results of agarose gel electrophoresis showed that except for the diffuse nucleic acid bands amplified from the leaves of lilies infected with LMoV, no nucleic acid bands were amplified in the other samples. This shows that the IC-RT-LAMP method established by the present invention has good specificity.

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Abstract

The invention discloses an IC-RT-LAMP kit used for specific detection of LiLy mottlt virus (LMoV), and a detection method thereof. The IC-RT-LAMP kit mainly comprises a specific LMoV antibody IgG, a specific RT reverse primer LMoV-R, a specific LAMP primer pair, a first chain cDNA synthesis reagent, an LAMP amplification reaction reagent, a fluorescent dye detection solution, a negative control, and a positive control; the specific LAMP primer pair is composed of a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primerLF, and a reverse loop primer LB; the negative control is ddH2O, the positive control is Lilium davidii LMoV CP gene standard substance. In the detection method, extraction of RNA is not needed, no molecular biology profession instrument such as enzyme-labeled instrument, PCR instrument, or fluorescent quantitation PCR instrument is needed, amplification reaction can be completed in constant temperature water bath, the specificity and the sensitivity are high, operation is convenient, the applicability is high, and the IC-RT-LAMP kit can be used in monitoring of infection, diffusion, and epidemic of lily LMoV, and is suitable for popularization.

Description

technical field [0001] The invention relates to an IC-RT-LAMP kit for specifically detecting lily mottle virus LMoV and a detection method thereof. Background technique [0002] Lily is an important economic crop that integrates ornamental, edible and medicinal uses. Long-term asexual reproduction leads to the accumulation of viruses, which seriously affects the yield and quality. Among them, Lily mottle virus (LMoV) is the most common virus. One is that it is widely distributed worldwide, especially in all temperate regions suitable for the growth of its host and in areas where aphid vectors are abundant. The latest field survey results of viral diseases showed that LMoV is also the main virus in edible lily in Lanzhou (Zhang et al., 2018). Viral diseases have seriously degraded the provenance of lily, which has become one of the important reasons restricting the production of lily and the export of cut flowers in my country. [0003] LMoV infection of Liliaceae plants of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6804C12Q1/6844C12R1/94
CPCC12Q1/6804C12Q1/6844C12Q1/701C12Q2563/131C12Q2521/107C12Q2531/119C12Q2563/107C12Q2565/125
Inventor 张玉宝王亚军谢忠奎杨果郭志鸿邱阳何玉惠
Owner NORTHWEST INST OF ECO ENVIRONMENT & RESOURCES CAS
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