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Primer group, kit and method for on-site constant-temperature visualization and fluorescence detection of cucumber green mottle mosaic virus

A green mottled flower and leaf virus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of high reagent cost, unsuitable on-site detection, poor stability of RPA technology, etc., to achieve Low equipment requirements, high sensitivity and good specificity

Pending Publication Date: 2021-08-06
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zeng et al. established a method for the detection of CGMMV by RPA technology. However, RPA technology has the problems of poor stability and high cost of reagents.
Li et al. established the RT-LAMP method to detect watermelon seed samples, but this method still requires an expensive fluorescent PCR instrument to detect the fluorescent signal to determine the detection result, and is still not suitable for on-site detection

Method used

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  • Primer group, kit and method for on-site constant-temperature visualization and fluorescence detection of cucumber green mottle mosaic virus
  • Primer group, kit and method for on-site constant-temperature visualization and fluorescence detection of cucumber green mottle mosaic virus
  • Primer group, kit and method for on-site constant-temperature visualization and fluorescence detection of cucumber green mottle mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Design and screening of primer set for constant temperature and visual detection of cucumber green mottle mosaic virus

[0046] 1. Primer set design

[0047] According to the whole genome sequence of cucumber green mottle mosaic virus isolate DY13 published on GenBank (accession number: KM873789.1), the highly conserved region was screened, and the primers were designed by LAMP Primer Explorer 5 software, and six sets of specific primers were screened and designed. A set of five to six primers, including two outer primers (OF and OB), two inner primers (IF and IB) and one or two loop primers (LB and / or LF), and using Primer blast ( http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) for evaluation. The gene sequences of the six sets of primers for cucumber green mottle mosaic virus designed in the present invention are shown in Table 1.

[0048] Table 1 Six sets of primers for on-site constant temperature and visual detection of cucumber green mottle mosaic vi...

Embodiment 2

[0060] The specific amplification verification of embodiment 2 primer set

[0061] 1. Preparation of positive control substance

[0062] According to the best sixth set of primers for cucumber green mottle mosaic virus obtained in Example 1, the specific constant temperature visual amplification gene sequence was selected to contain the genome sequence of cucumber green mottle mosaic virus such as SEQ ID NO.33. Cucumber green mottle mosaic virus positive control substance such as SEQ ID NO.33 was artificially synthesized to prepare a plasmid, which was used as a positive control (entrusted to Bao Biological Engineering (Dalian) Co., Ltd. to complete the preparation), aliquoted and stored at -20°C for future use.

[0063] 2. Specificity verification virus

[0064] In order to verify the specificity of the constant temperature visual detection of cucumber green mottle mosaic virus designed in the present invention, positive leaf samples infected by cucumber green mottle mosaic ...

Embodiment 3

[0089] Embodiment 3 constant temperature visual detection sensitivity test

[0090] 1. Sensitivity test method

[0091] 1.1 Optimization of primer set, 2× RNA constant temperature amplification reaction buffer and calcein dye concentration

[0092] The screened inner primers are incremented from 1.5 μM to 1.7 μM at a distance of 0.1 μM; the outer primers are incremented at a distance of 0.1 μM from 0.1 μM to 0.3 μM; the loop primers are incremented at a distance of 0.1 μM from 0.7 μM to 0.9 μM; 2 The ×RNA constant temperature amplification reaction buffer was increased from 0.75× to 1.25× at the interval of 0.25×; the concentration of 1× calcein dye was increased from 0.03× to 0.05× at the interval of 0.01×. The Taguchi method was used to explore the optimal combination of primer concentrations.

[0093] 1.2 Screening and optimization of Bst DNA polymerase

[0094] In order to determine the optimal dosage and ratio of Bst DNA polymerase, the optimal dosage was explored for ...

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Abstract

The invention belongs to the field of biological detection, and particularly relates to a primer group, a kit and a method for on-site rapid constant-temperature visualization and fluorescence detection of a cucumber green mottle mosaic virus. The primer group comprises an outer primer CGMMV-OF as shown in SEQ ID NO.1, an outer primer CGMMV-OB as shown in SEQ ID NO.2, an inner primer CGMMV-IF as shown in SEQ ID NO.3, an inner primer CGMMV-IB as shown in SEQ ID NO.4, a loop primer CGMMV-LF as shown in SEQ ID NO.5 and a loop primer CGMMV-LB as shown in SEQ ID NO.6. The primer group can be used for identifying the cucumber green mottle mosaic virus or detecting whether the cucumber green mottle mosaic virus exists in samples such as plant leaves, seeds, plants, nursery stocks, soil, field wastes and plant products (such as melons and fruits) or not. The detection method disclosed by the invention has the characteristics of simplicity in operation, rapidness, accuracy, good specificity, high sensitivity and result visualization, and is suitable for field and large-scale popularization and application.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a primer set, a kit and a method for on-site rapid constant temperature visualization and fluorescence detection of cucumber green mottle mosaic virus. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) belongs to the turnip mottle virus family, Tobamovirus genus. Virus particles are straight rods with a length of 300nm and a diameter of 18nm. The genome of CGMMV is a positive-sense single-stranded RNA with a total length of about 6.4kb. It has at least four open reading frames and encodes four proteins, corresponding to proteins of 186kb, 129kb, 29kb and 17.3kb, respectively. Among them, the 29kb protein is a mobile protein; the 17.3kb protein is a coat protein; the 186kb and 129kb proteins are enzymes necessary for virus replication and transcription. CGMMV is an important quarantine pest of cucurbit crops in many countries and regions in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2563/107C12Q2537/1376C12Q2521/107C12Q2545/113
Inventor 朴永哲尹新颖曹际娟郑秋月杨莉莉胡冰
Owner DALIAN NATIONALITIES UNIVERSITY
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