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Primers for detecting potato M viruses, kit and method

A detection method, potato technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems such as unclear functions

Inactive Publication Date: 2018-07-20
陈定虎
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ORFI of PVM RNA encodes a 223kDa replicase; ORF2, ORF3, and ORF4 encode a 25kDa, 12kDa, and 7kDa protein respectively, forming a three-gene cassette that participates in the intercellular movement of the virus; ORF5 encodes a 34kDa coat protein; overlapping ORF6 Encodes a cysteine-rich protein of 11-16kDa. The function of this product is unclear, but from the ability to bind nucleic acids, it may help aphids spread or be involved in host gene transcription and viral RNA replication[1]

Method used

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  • Primers for detecting potato M viruses, kit and method
  • Primers for detecting potato M viruses, kit and method
  • Primers for detecting potato M viruses, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] A kit for identifying potato M virus RT-LAMP, the kit includes:

[0078]

[0079] Among them, the primer sequence:

[0080] PVM-F3:5'-TATGAGGACCGGTTCGCC-3'

[0081] PVM-B3:5'-GACCATTCATACCGCCAGTC-3'

[0082] PVM-FIP:5'-CGGGGTTGGTCGCCTTATCAG-TTACGTGGAGAACACTGCTG-3'

[0083] PVM-BIP: 5'-AAGGACGTGGCTTTACGTGGT-ACCTCGGCATTAAGAGAGCT-3'.

Embodiment 2

[0085] A kit for identifying potato M virus RT-LAMP, the kit includes:

[0086]

[0087]

[0088] Among them, the primer sequence:

[0089] PVM-F3:5'-TATGAGGACCGGTTCGCC-3'

[0090] PVM-B3:5'-GACCATTCATACCGCCAGTC-3'

[0091] PVM-FIP:5'-CGGGGTTGGTCGCCTTATCAG-TTACGTGGAGAACACTGCTG-3'

[0092] PVM-BIP: 5'-AAGGACGTGGCTTTACGTGGT-ACCTCGGCATTAAGAGAGCT-3'.

[0093] The 2× reaction solution buffer described in the present invention is made up of following components:

[0094]

[0095] The enzyme solution is a mixture of Bst DNA polymerase and AMV reverse transcriptase.

[0096] The fluorescent visual detection reagent contains calcein fluorescent dye.

Embodiment 3

[0098] A method for detecting potato M virus, comprising the following steps:

[0099] A. Plant tissue total DNA extraction;

[0100] B. LAMP detection:

[0101] The kit described in Example 2 was used to configure the pre-reaction solution, and 5 μL of DNA of the sample to be detected was added to the pre-reaction solution to make the total amount reach 25 μL; the control group added 5 μL of deionized water to the pre-reaction solution; positive control Add 5 μL of virus-carrying DNA to the pre-reaction solution; use a pipette to mix by aspiration and drainage, or close the lid and tap lightly to make the solution fully mixed and then centrifuge instantaneously, avoiding bubbles during mixing; place the mixture in the reaction well, and React at a constant temperature of 65°C for 90 minutes, and finally keep it at 80°C for 5 minutes to end the reaction;

[0102] C. LAMP result judgment

[0103] After the amplification reaction is over, use an ultraviolet irradiation device...

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Abstract

The invention discloses primers for detecting potato M viruses, a kit and a method, aims at providing the primers which are quick, simple, efficient and high in accuracy, specificity and sensitivity and are for detecting potato X viruses RT-LAMP, also aims at providing the kit which comprises the primers and is used for detecting the potato M viruses and also aims at providing the detection methodof the potato M viruses RT-LAMP. The primers comprise PVM-F3, PVM-B3, PVM-FIP and PVM-BIP.

Description

technical field [0001] The invention relates to a primer, a kit and a detection method for identifying potato M virus RT-LAMP, and belongs to the field of biotechnology. Background technique [0002] Potato virus M, PVM was found in the United States, France, the United Kingdom, Germany, the Netherlands and other countries. At present, PVM occurs all over the world [1]. PVM is a member of Carlavirus genus Carlavirus. The size of the virion is 650nmX12nm, and there is an incompletely closed viral capsid. The lethal temperature is 65-71°C, the in vitro preservation period is 5-7 days, and the dilution limit is 10 2 -10 3 . The virus could be detected in any part of the host plant, and the virus mainly existed in the cytoplasm. Amorphous X-body-shaped inclusion bodies were found in the cytoplasm of susceptible plant cells. The virus genome is a single-stranded positive-sense RNA with a length of 8535bp. There is a Poly(A) at the 3' end and a methylated cap structure at th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107C12Q2521/107
Inventor 陈定虎
Owner 陈定虎
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