RT-RAA-LFS rapid visual detection primer, probe and kit for tomato mosaic virus, and applications of RT-RAA-LFS rapid visual detection primer, probe and kit

A technique for detecting tomato mosaic virus and primers, which is applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as high cost, heavy preparatory work, and strong dependence, and achieve Low equipment requirements, high sensitivity, and universal applicability

Pending Publication Date: 2021-09-24
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disadvantage of this method is that it is highly dependent on laboratory equipment, requires a certain professional background, and the preparatory work for this detection method is heavy and the cost is high.

Method used

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  • RT-RAA-LFS rapid visual detection primer, probe and kit for tomato mosaic virus, and applications of RT-RAA-LFS rapid visual detection primer, probe and kit
  • RT-RAA-LFS rapid visual detection primer, probe and kit for tomato mosaic virus, and applications of RT-RAA-LFS rapid visual detection primer, probe and kit
  • RT-RAA-LFS rapid visual detection primer, probe and kit for tomato mosaic virus, and applications of RT-RAA-LFS rapid visual detection primer, probe and kit

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0027] 1. Materials

[0028]Plants to be tested: hot pepper (Hangjiao No. 1 resistant variety), Shidun 197 large tomato seeds were planted in a constant temperature greenhouse in the laboratory, the cultivation conditions were 16 hours of light, 8 hours of darkness, and a constant temperature of 22 °C; The plants were collected from Yunnan, Ningxia and Zhejiang provinces; 8 tomato plants with suspected virus disease samples were collected from Shandong and Zhejiang provinces; the pepper plant disease samples infected by PMMoV and TSWV were preserved in our laboratory. All plant samples were frozen in liquid nitrogen and stored at -70 °C.

[0029] Tested strains and vectors: Agrobacterium strains carrying ToMV invasive clones (Pokl4 2-ToMV) were kept in the laboratory.

[0030] Reagent consumables: RNA constant temperature rapid amplification kit (colloidal gold test strip type) was purchased from Weifang Anpu Future Biotechnology Company; T7 in vitro transcription kit was pur...

specific Embodiment 2

[0043] 1. Preliminary screening of ToMV-RAA primer combinations

[0044] Take ToMV and TMV as the same Tobamovirus Genus, the genome sequences of the two are highly homologous (the homology is about 89.6% after comparison), and the ToMV virus isolates are also highly conserved. Therefore, six genome sequences of ToMV and TMV virus isolates were downloaded from the database of the National Center for Biotechnology Information (NCBI) in the United States, and DNAMAN 8.0 was used for multiple sequence alignment. Through comparison, 8 pairs of primers were designed for RAA-AGE primary screening in the relatively low homology between ToMV genome sequence and TMV motor protein (MP) and coat protein (CP). The design principles are as follows:

[0045] (1) In order to improve the sensitivity of the RAA reaction, the length of the upstream and downstream primers should be between 30 and 35 bp;

[0046] (2) The secondary structure of the downstream primers, in principle, requires no ...

specific Embodiment 3

[0076] Sensitivity assessment of ToMV-(RT)RAA-LFS system

[0077] 1. Use the pEASY-ToMV-MP&CP recombinant plasmid as a template

[0078] Use ddH to pEASY-ToMV-MP&CP recombinant plasmid 2 O sequentially (10 6 ~10 0 copy / μL) ten-fold serial dilution, ddH 2 O is a negative control template; prepare pepper, tomato healthy leaf mixed sample crude extract, take each 300 mg of healthy leaves and place them in a grid grinding sample bag, add 2 mL of PBS solution containing 0.05% Tween-20, fully grind, and then The pEASY-ToMV-MP&CP recombinant plasmid was carried out sequentially with the crude extracts of pepper and tomato healthy leaf mixture (10 6 ~10 0 copy / μL), the crude extract of healthy leaves was used as a negative control template, and the above-diluted template was placed on ice for future use.

[0079] Using the above two different diluted templates, the optimal primer probes screened out were used as the primer probe combination for the subsequent evaluation of the...

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Abstract

The invention discloses a RT-RAA-LFS rapid visual detection primer, probe and kit for tomato mosaic virus and applications of the RT-RAA-LFS rapid visual detection primer, probe and kit. The RT-RAA-LFS rapid visual detection primer, probe and kit are characterized in that the specific primers and the fluorescent probes are designed according to motion protein and coat protein segments of the tomato mosaic virus, and the primer sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; and the kit comprises the following reaction systems of 29.4 [mu]L of a buffer solution A, 2.1 [mu]L of upstream primer of 10 [mu]M, 3 [mu]L of downstream primer of 10 [mu]M, 0.6 [mu]L of probe of 10 [mu]M, 1 [mu]L of RNA plant juice crude extract template, 11.4 [mu]L of ddH2O and 2.5 [mu]L of buffer solution B, and the total volume is 50 [mu]L. The RT-RAA-LFS rapid visual detection primer, probe and kit have the advantages of high sensitivity, high specificity and rapid and accurate detection.

Description

technical field [0001] The invention relates to the detection of tomato mosaic virus, in particular to RT-RAA-LFS rapid visual detection primers, probes, kits and applications of tomato mosaic virus. Background technique [0002] Tomato mosaic virus (ToMV) is Virgaviridae division Tobamovirus Positive-strand RNA viruses of the genus. Because the nucleic acid sequence of the virus is highly homologous to tobacco mosaic virus (TMV), researchers have reported ToMV as a branch of TMV for a long time. It was not until 1971 that ToMV was distinguished from TMV and isolated as an independent virus species. ToMV can infect plants such as Solanaceae, Brassicaceae, Gramineae, Chenopodiaceae, and Fabaceae, among which pepper is one of the natural hosts of ToMV. Peppers infected by ToMV usually produce local lesions, causing systemic necrosis of plants, which seriously affect the quality and yield of peppers. In addition, in recent years, ToMV has also caused reports of viral dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12N15/11C12R1/94
CPCC12Q1/701C12Q1/6844C12Q1/6804C12Q2521/507C12Q2531/119C12Q2563/107C12Q2563/131C12Q2565/625
Inventor 郑红英来宇超宋雪梅曹宇浩彭杰军鲁宇文陈剑平燕飞
Owner NINGBO UNIV
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