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A kind of broad-spectrum detection kit of cowpea mosaic virus subfamily virus and method thereof

A cowpea mosaic virus and kit technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/testing, etc., can solve the problems of no serology, no molecular detection methods, etc., and achieve high sensitivity, The effect of short detection cycle and rapid detection and monitoring

Active Publication Date: 2019-07-02
INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, within the Vigomovirus subfamily, most virus species do not have corresponding serological and rapid molecular assays

Method used

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  • A kind of broad-spectrum detection kit of cowpea mosaic virus subfamily virus and method thereof
  • A kind of broad-spectrum detection kit of cowpea mosaic virus subfamily virus and method thereof
  • A kind of broad-spectrum detection kit of cowpea mosaic virus subfamily virus and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, broad-spectrum detection kit of cowpea mosaic virus subfamily virus and detection method thereof

[0085] 1. Screening of the first round of primers for detection of cowpea mosaic virus subfamily virus

[0086] 1. Extraction of RNA

[0087] The samples containing Arabidopsis mosaic virus (Agdia LPC 23203(C1954)), faba bean wilt virus No. C1319)), cowpea heavy mosaic virus (ATCC PV-273), rubus ringspot virus (DSMZ PV-0429), radish mosaic virus (DSMZ PV-0355) and squash mosaic virus (Agdia LPC 26400 (C2013) ) Total RNA of diseased leaf samples.

[0088] 2. Reverse transcription reaction

[0089] In a 0.6mL centrifuge tube, add 4 μL of total RNA of the sample to be tested, 2 μL of primer SecoV-Rat (10 μmol / L), 8 μL of DEPC-treated ddH2O, and put it in a water bath at 95°C for 10 minutes, then quickly place it on ice for 5 minutes. Immediately, continue to add 4 μL of 5×M-MLV reverse transcription buffer, 1 μL of dNTPs (10 mmol / L), 0.5 μL of M-MLV reverse ...

Embodiment 2

[0135] Example 2, broad-spectrum and specificity detection of Cowpea mosaic virus subfamily virus broad-spectrum detection kit

[0136] 1. Broad-spectrum and specificity detection of the broad-spectrum detection kit for cowpea mosaic virus subfamily virus

[0137] 1. Extract Andes potato mottle virus (DSMZ PV-0057), Arabidopsis mosaic virus (Agdia LPC 23203 (C1954)) and faba bean true mosaic virus (DSMZ PV-0098) containing Cowpea mosaic virus subfamily respectively , Broad Bean Wilt Virus (ACD V012-P1), Broad Bean Wilt Virus No. 1 (DSMZ PV-0067), Broad Bean Wilt Virus No. 2 (ACD V212-P1), Broad Bean Wilt Virus No. 2 (DSMZ PV-0862), Broad Bean Wilt Virus No. 1 & 2 (Agdia LPC 46202 (C1543)), broad bean wilt virus No. 1 & 2 (Agdia LPC 46202 (C1643)), blueberry leaf spot virus (Agdia LPC 88001 (C1796)), bean pod mottle virus (ACD V082-P1), vegetable Pod mottle virus (Agdia LPC 46400(C1858)), cowpea mosaic virus (Agdia LPC 24200(C1319)), cowpea heavy mosaic virus (ATCC PV-273), gr...

Embodiment 3

[0145] Example 3, Sensitivity Detection of Cowpea Mosaic Virus Subfamily Virus Broad Spectrum Detection Kit

[0146] 1. Extract the total RNA of the diseased leaf sample containing squash mosaic virus (Agdia LPC 26400 (C2013)).

[0147] 2. Synthesize the cDNA template according to the method of 1 in step 3 of Example 1, and carry out 10-fold serial dilution of the synthesized cDNA template into 10 -1 ~10 -10 A total of 10 cDNA templates with gradient concentrations were detected according to the kit and detection method in Example 1 to verify the sensitivity of the kit. Each sample was tested in triplicate.

[0148] Test results such as Figure 4 shown. The results show that the detection kit of the present invention can detect 10 -8 The concentration of the cDNA template shows that the primers of the present invention have high sensitivity, which is sufficient to meet the needs of daily detection.

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Abstract

The invention discloses a comovirinae virus broad-spectrum detection kit and a detection method thereof. The comovirinae virus broad-spectrum detection kit comprises a set of primers consisting of three primers. The experiment proves that the set of primers for detecting comovirinae virus and the detection kit have the characteristics of good broad spectrum, high sensibility, strong specificity, short detection period and the like, and is suitable for agricultural production and rapid detection and monitoring of comovirinae virus on the port of entry and exit.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, and relates to a broad-spectrum detection kit for cowpea mosaicinae virus and a detection method thereof, in particular to a set of primers, a kit and a detection method for cowpea mosaicinae virus. Background technique [0002] The subfamily Comovirinae belongs to the order Picornavirales and the family Secoviridae, including the genus Comovirus, Fabavirus and Nepovirus. ), containing 15, 5 and 36 viruses, respectively. Among the cowpea mosaic viruses, 11 viruses have a relatively narrow host range and only infect leguminous plants, while other species infect plants such as Solanaceae, Cruciferae, and Cucurbitaceae. Cowpea mosaic virus mainly causes mosaic and mottled symptoms, and usually does not cause ring spot symptoms. Viruses of the fabavirus genus have a very broad host range, including dicotyledonous plants, as well as some families of monocotyledonous plants. Symptoms caused...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/94
CPCC12Q1/701
Inventor 廖富荣沈建国黄蓬英方志鹏林振基
Owner INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU