Kit for detection of arabis mosaic virus (ArMV) of lilies and detection method of arabis mosaic virus

A technology of A. lily thaliana and detection methods, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as high technical requirements, improve detection efficiency, high sensitivity, and reduce experiment difficulty Effect

Pending Publication Date: 2018-11-06
NORTHWEST INST OF ECO ENVIRONMENT & RESOURCES CAS
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  • 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 ArMV virus detection method that require high technical requirements and rely on professional instruments and equipment, and provide a method that can specifically and efficiently detect lily and Arabidopsis thaliana flowers and leaves without professional molecular biology instruments and equipment. The IC-RT-LAMP method for viruses; specifically, the combination of immunology and LAMP technology is ingenious. First, the specific antibody of the ArMV virus is coated on the wall of the coating container to specifically capture the ArMV particles in the detection sample, thereby To concentrate and purify the virus, and then use the captured ArMV particles as a template to carry out reverse transcription RT and then LAMP reaction to achieve the purpose of detecting lily and A. thaliana mosaic virus

Method used

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  • Kit for detection of arabis mosaic virus (ArMV) of lilies and detection method of arabis mosaic virus
  • Kit for detection of arabis mosaic virus (ArMV) of lilies and detection method of arabis mosaic virus
  • Kit for detection of arabis mosaic virus (ArMV) of lilies and detection method of arabis mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment １

[0052] Example 1: The acquisition of positive control substance

[0053] 1. Extraction of total RNA

[0054] Grind 50-100 mg of ArMV-infected cut lily Sorbonne leaves in liquid nitrogen, and extract total RNA from susceptible tissues with a plant total RNA extraction kit.

[0055] 2. Primer design and synthesis

[0056] A pair of specific forward (ArMV-F) and reverse primers (ArMV-R) were designed according to the sequence of the lily ArMV CP gene registered on GenBank (GenBank accession number: AB279740.2), and the sequences of the above primers are as follows:

[0057] ArMV-F: 5'-TACTACCAGTGCCTACAAA-3';

[0058] ArMV-R: 5'-AATCATTAGGGAAAGTCG-3'.

[0059] 3. Preparation of positive control substance

[0060] 1) RT reaction

[0061] Using ArMV reverse primers ArMV-R and M-MLV reverse transcriptase in cut lily lily to carry out RT reaction to synthesize the first strand of cDNA.

[0062] The 10 μL RT reaction system is as follows: 2 μL total RNA, 10 μM ArMV-specific rever...

Embodiment ２

[0072] Example 2: Establishment of an immunocapture IC-RT-LAMP method for detection of Lily ArMV

[0073] 1, the preparation method of rabbit anti-ArMV polyclonal antibody IgG among the present invention:

[0074] 1) Preparation of ArMV CP genetically engineered recombinant protein: extract total RNA from the leaves of cut lily Sorbonne infected with ArMV and perform RT-PCR to amplify the CP gene fragment of ArMV; clone into pET-28a vector by double enzyme digestion; recombinant Plasmid transformed into E. coli BL21 (DE3), cultured with shaking at 37°C, induced expression with IPTG, and purified on Ni-NTA column to obtain a highly pure ArMV CP genetically engineered recombinant protein with a size of 55.0 kDa.

[0075] 2) Preparation of polyclonal IgG: New Zealand white rabbits were immunized with the above-mentioned purified 1 mg / mL ArMV CP genetically engineered recombinant protein as an immunogen.

[0076] In the first immunization, the protein antigen and Freund's compl...

Embodiment ３

[0096] Example 3: Specificity of detection of ArMV by immunocapture IC-RT-LAMP

[0097] In order to analyze the specificity of immunocapture IC-RT-LAMP in detecting ArMV, the three main viruses (LSV, CMV, LMoV) that most commonly infect lily and the susceptible leaves of ArMV were used as samples, and the ArMV polyclonal antibody IgG was used to capture the virus respectively. Particles were 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.

[0098] The results of agarose gel electrophoresis showed that except for the diffuse nucleic acid bands amplified in the leaves of lilies infected with ArMV, 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

Disclosed is a kit for detection of IC-RT-LAMP of arabis mosaic virus (ArMV) of lilies and a detection method of the arabis mosaic virus and a detection method of the arabis mosaic virus. The kit mainly comprises a specific ArMV antibody IgG, a specific RT reverse primer ArMV-R, a specific LAMP primer group, a first chain cDNA synthesis reagent, an LAMP amplification reaction reagent, fluorescentdye detection fluid, a negative reference substance and a positive reference substance; the specific LAMP primer group is formed by a positive external primer F3, a reverse external primer B3, a positive internal primer FIP, a reverser internal primer BIP and a positive loop primer LF; the negative reference substance is ddH2O, and the positive reference substance is an ArMV gene standard substance for cut-flower lilies. Extraction of RNA (ribonucleic acid) is omitted, professional instruments and equipment for molecular biology of microplate readers, PCR (polymerase chain reaction) instruments are not needed, amplified reaction can be completed in thermostatic waterbath, and the kit is high in specificity and sensitivity, convenient in operation, high in applicability, applicable to monitoring of the occurrence, spread and prevalence of the ArMV virus in the lilies, and suitable for grassroots application.

Description

technical field [0001] The invention relates to an IC-RT-LAMP kit for specifically detecting Arabidopsis mosaic virus ArMV infecting lilies 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 causes the accumulation of viruses, which seriously affects the yield and quality. important reason for export. At present, there are more than 20 kinds of viruses reported in the literature to infect lily, except lily mottled virus ( Lily mottle virus , LMoV), cucumber mosaic virus ( Cucumber mosaic viru s, CMV) and lily recessive virus ( Lily symptomless virus , LSV), Arabidopsis mosaic virus ( Arabis mosaic virus , ArMV) is also one of them (Kulshrestha et al., 2005). In 2017, in a field virus survey, we detected ArMV virus for the first time in a cut lily cultivar Sorbonne grown in Gansu. Symptoms of ArMV infestation of lilies mainly include m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107
Inventor 张玉宝王亚军谢忠奎杨果邱阳郭志鸿王乐
Owner NORTHWEST INST OF ECO ENVIRONMENT & RESOURCES CAS
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