IC-RT-LAMP kit for detecting ToMV virus infecting angelica and detecting method thereof

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 complex operation, expensive equipment, and high technical level of operators, and achieve The effect of simplifying the operation process, reducing the difficulty of detection, and reducing the difficulty of experiment

Inactive Publication Date: 2019-05-17
NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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...

Method used

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  • IC-RT-LAMP kit for detecting ToMV virus infecting angelica and detecting method thereof
  • IC-RT-LAMP kit for detecting ToMV virus infecting angelica and detecting method thereof
  • IC-RT-LAMP kit for detecting ToMV virus infecting angelica and detecting method thereof

Examples

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 kit for specifically detecting tomato mosaic virus ToMV infecting angelica and a detecting method thereof. The kit mainly comprises a ToMV antibody IgG, an RT reverse primer ToMV-R, an LAMP primer set, a first strand cDNA synthesis reagent, an LAMP amplification reaction reagent, a fluorescent dye detection solution, a negative control and a positive control.The kit is high in specificity with the sensitivity reaching 0.45pg/mL which is 100 times higher than ordinary PCR. No need to extract RNA exists, no special instrument is required, the operation process is simplified, the detecting difficulty is reduced, and the detecting efficiency is improved. The kit provides a new technology platform for detecting the ToMV, can be used for monitoring occurrence, spreading and prevalence of the Chinese herbal medicine angelica and is suitable for promotion and application at the primary level.

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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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12N15/11
Inventor 张玉宝王若愚谢忠奎张和平王筠赵霞周琴
Owner NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
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