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IC-RT-LAMP detection method for specific detection of angelica sinensis ToMV viruses

A technology of IC-RT-LAMP and detection methods, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high technical level requirements for operators, complex operations, expensive instruments and equipment, etc. Achieve the effect of reducing the difficulty of experiments, simplifying the operation process and reducing the difficulty of detection

Inactive Publication Date: 2019-05-03
NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] Purpose of the invention: In view of the complex operation of the ToMV detection method of tomato mosaic virus in the prior art, high requirements on the technical level of operators, and expensive equipment, the purpose of the invention is to provide a tomato mosaic virus that infects Angelica sinensis The IC-RT-LAMP specific detection primer composition of ToMV; Another object of the present invention is to provide the IC-RT-LAMP detection kit of above-mentioned tomato mosaic virus ToMV; Another object of the present invention is to provide above-mentioned tomato mosaic IC-RT-LAMP detection method of virus ToMV

Method used

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  • IC-RT-LAMP detection method for specific detection of angelica sinensis ToMV viruses
  • IC-RT-LAMP detection method for specific detection of angelica sinensis ToMV viruses
  • IC-RT-LAMP detection method for specific detection of angelica sinensis ToMV viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the acquisition of positive control substance

[0061] 1. Extraction of total RNA

[0062] Grind 50-100 mg of ToMV-infected leaves of Minxian Angelica sinensis in liquid nitrogen, and extract total RNA from susceptible tissues with a plant total RNA extraction kit;

[0063] 2. Primer design and synthesis

[0064] A pair of specific forward (ToMV-F) and reverse primers (ToMV-R) were designed and synthesized according to the Angelica ToMV CP gene sequence registered on GenBank (GenBank accession number: AB355139.1); the sequences of the above primers are as follows:

[0065] ToMV-F: 5'-TACTCAATCACTTCTCCATCGC-3'

[0066] ToMV-R: 5'-GATGCAGGTGCAGAGGTCCAG-3';

[0067] 3. Preparation of positive control substance

[0068] 1) RT reaction

[0069] Use Angelica ToMV reverse primer ToMV-R and M-MLV reverse transcriptase to carry out RT reaction to synthesize the first strand of cDNA;

[0070] The 10 μL RT reaction system is as follows: 2 μL total RNA, 10 μM To...

Embodiment 2

[0080] Embodiment 2: A kind of IC-RT-LAMP detection kit for specific detection Angelica ToMV

[0081] The kit consists of ToMV antibody IgG, antibody coating buffer (CB), phosphate buffer (PBS), phosphate wash buffer (PBST), RT reverse primer ToMV-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 and reverse inner primer The sequences of BIP, the above-mentioned specific RT reverse primer ToMV-R and the LAMP primer set are as follows:

[0082] ToMV-R: 5'-GATGCAGGTGCAGAGGTCCAG-3';

[0083] F3: 5'-TTTTAGATCCTCTAATTACTGCG-3';

[0084] B3: 5'-CCAACCCAGACATACTTTCA-3';

[0085] FIP: 5'-GTCGGACTCTGCTGGTTTTTTTATTGCTGGGGACTTTCGATA-3';

[0086] BIP: 5'-GGTAGACGACGCTACGGTTGTCCAGTACCTCTTACTAGTTCAT-3';

[0087] First-strand cDNA...

Embodiment 3

[0091] Example 3: Establishment of the method for detecting Angelica ToMV by immune capture IC-RT-LAMP

[0092] 1, the preparation method of rabbit anti-ToMV polyclonal antibody IgG among the present invention:

[0093] 1) Expression and purification of ToMV CP genetic engineering fusion protein: extract total RNA from the leaves of Minxian Angelica sinensis infected with ToMV, perform reverse transcription polymerase chain reaction (RT-PCR), and amplify the CP gene fragment of ToMV; Excised and cloned into the pET-28a vector; 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 ToMV CP genetic engineering fusion protein with a size of 17.6kDa;

[0094] 2) Preparation of polyclonal antibody IgG: New Zealand white rabbits were immunized with 1 mg / mL of the above-mentioned ToMV CP genetically engineered fusion protein as an immunogen;

[0095] In the first immunizat...

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Abstract

The invention discloses an IC-RT-LAMP detection method for specific detection of angelica sinensis ToMV viruses. The IC-RT-LAMP detection method comprises the main steps of performing specific capturing on ToMV virions by using antibody IgG, performing inverse transcription and an RT reaction synthesis through a ToMV specific primer to obtain cDNA, then performing LAMP amplification with a specific LAMP primer, after a reaction is finished, detecting whether samples to be detected contain the ToMV viruses or not by using a fluorochrome visual study method or an agarose gel electrophoresis method. The IC-RT-LAMP detection method is high in specificity, the sensitivity can achieve 0.45pg / mL and is 100 times higher than that of common PCR, extraction of RNA is not needed, and professional instrument or equipment is not needed, so that the operation processes are simplified, the detection difficulty is reduced, the detection efficiency is improved, and a technique platform is provided fordetection of angelica sinensis ToMV. The IC-RT-LAMP detection method can be used for monitoring the generation, the diffusion and the propagation of the angelica sinensis ToMV viruses, and is suitablefor promotion and application in primary levels.

Description

technical field [0001] The invention relates to an IC-RT-LAMP kit for specifically detecting tomato mosaic virus infecting angelica and a detection method thereof. Background technique [0002] Angelica sinensis, also known as Min Angelica, Dry Gui, Horsetail Angelica, Horsetail Gui, Yungui, West Angelica, Golden Angelica, Earth Angelica, is a perennial herb of Umbelliferae. Its roots are often used as medicine. , the effect of moistening the intestines; containing basic lactone (ligusiilide), n-butylidene phthalide (n-butylidene phthalide), ferulic acid, vitamin B12, vitamin E, niacin, sucrose and various amino acids, and sesquiterpenes compounds, etc. Due to the unique ingredients, its medicinal value is very high, and there is a saying of "ten directions and nine returns". In addition to nourishing blood, promoting blood circulation, regulating menstruation and relieving pain, moistening dryness and laxative, it also has calming brain, skin care, reducing pigmentation, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
Inventor 张玉宝王若愚谢忠奎王筠赵霞周琴
Owner NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
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