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A broad-spectrum detection kit for cowpea mosaic virus and its detection method

A cowpea mosaic virus and kit technology, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, can solve the problem of broad-spectrum molecular detection methods without cowpea mosaic virus, and no Cowpea mosaic virus detection kit and other problems, to achieve the effect of short detection period, high sensitivity and good broad-spectrum

Active Publication Date: 2016-04-20
厦门佰能检验技术服务有限公司
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  • 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

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  • A broad-spectrum detection kit for cowpea mosaic virus and its detection method
  • A broad-spectrum detection kit for cowpea mosaic virus and its detection method
  • A broad-spectrum detection kit for cowpea mosaic virus and its detection method

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) TaqDNA 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 f...

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] Andean potato mottle virus (DSMZPV-0057), faba bean true mosaic virus (DSMZPV-0098), bean pod mottle virus (ACDV082-PV), bean pod mottle virus (AgdiaC1858) and cowpea mosaic virus (AgdiaC1319) were extracted respectively. ), cowpea mosaic virus (AgdiaC1860), cowpea heavy mosaic virus (ATCCPV-273), cowpea heavy mosaic virus (DSMZPV-0050), radish mosaic virus (DSMZPV-0355), pumpkin mosaic virus (AgdiaC2013), etc. The total RNA of the diseased leaf samples of 10 isolates of 7 kinds of viruses was detected by the kit of Example 1 and the method of Example 2, and the broad-spectrum of the kit was verified. 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 iso...

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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

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Application Information

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Patent Type & Authority Patents(China)
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