Unlock instant, AI-driven research and patent intelligence for your innovation.

Broad-spectrum detection kit and detection method of Comovirus

The technology of a cowpea mosaic virus and a kit is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, and can solve the problem of no cowpea mosaic virus detection kit and no cowpea mosaic virus detection kit. Broad-spectrum molecular detection methods for virus genus and other problems, to achieve the effect of short detection cycle, high sensitivity and good broad-spectrum

Active Publication Date: 2014-12-17
厦门佰能检验技术服务有限公司
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no broad-spectrum molecular detection method for cowpea mosaic virus, and there is no detection kit specifically for the broad spectrum of cowpea mosaic virus

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Broad-spectrum detection kit and detection method of Comovirus
  • Broad-spectrum detection kit and detection method of Comovirus
  • Broad-spectrum detection kit and detection method of Comovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, cowpea mosaic virus genus virus broad-spectrum detection kit configuration (50 times detection amount)

[0060] 1) Primer Oligo(dT) 18 : The concentration is 10μmol / L, 1 tube (100μL);

[0061] 2) Reverse transcription buffer: 5×, 1 tube (250 μL);

[0062] 3) dNTPs: the concentration is 10mmol / L, 1 tube (100μL);

[0063] 4) M-MLV reverse transcriptase: 200U / μL, 1 tube (30μL);

[0064] 5) RNase inhibitor: 40U / μL, 1 tube (30μL);

[0065] 6) RNase-free ddH 2 O, 1 tube (1 mL);

[0066] 7) PCR buffer (Mg containing 25mmol / L 2+ ): 10×, 1 tube (300μL);

[0067]8) Taq DNA polymerase: 5U / μL, 1 tube (30μL);

[0068] 9) Primer ComoV-2-F2-M4: the concentration is 1 μmol / L, 1 tube (120 μL);

[0069] 10) Primer ComoV-2-R2-M1: the concentration is 1 μmol / L, 1 tube (120 μL);

[0070] 11) Primer M4: the concentration is 10 μmol / L, 1 tube (120 μL);

[0071] 12) Primer M1: the concentration is 10 μmol / L, 1 tube (120 μL);

[0072] 13) Positive control sample: leaf ...

Embodiment 2

[0086] Embodiment 2, the detection method of cowpea mosaic virus broad-spectrum detection kit

[0087] The detection method of the cowpea mosaic virus broad-spectrum detection kit in embodiment 1 may further comprise the steps:

[0088] 1) Reverse transcription reaction: In the PCR tube, add 4 μL of total RNA (extracted by Trizol method) of the sample to be tested, 2 μL of primer Oligo(dT) with a concentration of 10 μmol / L 18 , 8 μL RNase-free ddH 2 O, after 10 min in water bath at 65°C, quickly put it on ice for 5 min, continue to add 4 μL of 5× reverse transcription buffer, 1 μL of dNTPs with a concentration of 10 mmol / L, and 0.5 μL of M-MLV with a concentration of 200 U / μL for inversion Recording enzyme, 0.5 μL of RNase inhibitor with a concentration of 40 U / μL, reacted in a water bath at 42°C for 1 hour, then cooled to room temperature naturally in a water bath at 70°C for 15 minutes (to inactivate reverse transcriptase), and synthesized cDNA.

[0089] 2) PCR reaction: 2...

Embodiment 3

[0091] Embodiment 3, the broad-spectrum detection of cowpea mosaic virus broad-spectrum detection kit

[0092] Extracts containing Andean potato mottle virus (DSMZ PV-0057), faba bean true mosaic virus (DSMZ PV-0098), bean pod mottle virus (ACD V082-PV), bean pod mottle virus (Agdia C1858), cowpea flower Leaf virus (Agdia C1319), cowpea mosaic virus (Agdia C1860), cowpea heavy mosaic virus (ATCC PV-273), cowpea heavy mosaic virus (DSMZ PV-0050), radish mosaic virus (DSMZ PV-0355) , pumpkin mosaic virus (Agdia C2013) and other 7 kinds of virus 10 isolates diseased leaf samples of the total RNA, utilize the method of embodiment 1 kit and embodiment 2 to detect, verify the broad-spectrum of this kit. Each sample was tested in triplicate.

[0093] At the same time, a blank control with water instead of the PCR reaction template was set up in the experiment.

[0094] The results showed that the bands of about 600bp were detected in the diseased leaf samples of 10 isolates of 7 vi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a broad-spectrum detection kit and a detection method of Comovirus. The broad-spectrum detection kit provided by the invention contains a primer set constituted by a reverse transcription primer and four amplification primers, wherein the reverse transcription primer is a single-stranded DNA as shown in sequence 1 in a sequence table; the four amplification primers are four single-stranded DNA as shown in sequence 2, sequence 3, sequence 4 and sequence 5 in the sequence table. Experiments prove that the primer set for detecting Comovirus and the kit thereof provided by the invention have the characteristics of good broad-spectrum property, high sensitivity, strong specificity, short detection period and the like and are suitable for fast detection and monitoring of the Comovirus in ports of entry and exit and agricultural production.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, and relates to a broad-spectrum detection kit for cowpea mosaic virus and a detection method thereof, in particular to a primer set, a kit and a detection method for detecting cowpea mosaic virus. Background technique [0002] Comovirus is one of the eight virus genera in the order Picornavirales and the associated Secoviridae, and it is related to Fabavirus, Nepovirus Comovirinae consists of 15 virus members and 1 tentative species. The host range of this type of virus is relatively narrow. Among the 15 kinds of cowpea mosaic viruses that have been found, 11 viruses only infect leguminous plants, such as soybeans, cowpeas, broad beans, etc.; 3 viruses are respectively harmful to Solanaceae, Brassicaceae, Cucurbitaceae and other plants; the other two species are harmful to red clover and root tubers respectively. Mosaic and mottling are the main symptomatic characteristics of the virus...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701
Inventor 廖富荣方志鹏林振基吴媛
Owner 厦门佰能检验技术服务有限公司