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Primer and kit for using RPA for detecting procambarus clarki picornaviridae and detection method

A technology for RNA virus and Crayfish is applied in the field of special primers for detection, which can solve the problems of low timeliness, long time, complicated and tedious operations, etc., and achieve the effects of high sensitivity and strong specificity

Inactive Publication Date: 2019-05-10
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods require expensive PCR machines or special equipment and professional test sites. At the same time, the process of PCR amplification requires special thermal cycle equipment, low-temperature storage reagents and technical operation requirements to avoid cross-contamination, and the operation is complicated. It is cumbersome, time-consuming, and has low timeliness. At the same time, it also needs to have a certain molecular biological basis and operational technical diagnosis requirements, which limits the application of PCR in on-site diagnosis.

Method used

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  • Primer and kit for using RPA for detecting procambarus clarki picornaviridae and detection method
  • Primer and kit for using RPA for detecting procambarus clarki picornaviridae and detection method
  • Primer and kit for using RPA for detecting procambarus clarki picornaviridae and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] According to the sequence of Procambarus clarkii picornavirus, an RPA primer pair consisting of upstream and downstream primers with the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 was designed.

[0038] Upstream primer RPA PcPV-1F: 5'-CTGATGAGGAATTAGACGATCTTATCGTTG-3' (SEQ ID NO.1);

[0039] Downstream primer RPA PcPV-1R: 5'-CAGAATTAAAGACCGACGTTAGTAGATGAC-3' (SEQ ID NO.2).

Embodiment 2

[0041] Using the TRIzol method, viral RNA was extracted from Procambarus clarkii known to be infected with picornaviruses and reverse transcribed into cDNA. The specific operation process is as follows.

[0042]Take an appropriate amount (0.02g-0.05g) of the hepatopancreas of Procambarus clarkii into an EP tube, and put small magnetic beads in the tube (2 in each EP tube), add 1ml of RNA Plus, and put it into the homogenate In the machine for 2-3 minutes, after the end, let it stand for 5 minutes.

[0043] Put the sample into a refrigerated centrifuge at 4°C, centrifuge at 12000rpm for 5min, and take 600μL of supernatant.

[0044] Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes.

[0045] Centrifuge at 12000rpm for 15min at 4°C, take 200μL of the supernatant,

[0046] Add 500 μL of 100% isopropanol, invert up and down 5 times to mix well, and place at room temperature for 10 minutes.

[0047] Centrifuge at 12000rp...

Embodiment 3

[0053] Using the cDNA obtained in Example 2 as a template and the RPA primers designed in Example 1, the RPA reaction was carried out in the following reaction system.

[0054] Sample detection: Prepare 47.5 μl of mixed reaction solution, including 29.5 μl Rehydration Buffer, 2.4 μl of 10 μM upstream primer, 2.4 μl of 10 μM downstream primer, 1 μl of template cDNA and ddH 2 O 12.5 μl. The mixed solution is mixed and transferred to the reaction unit tube containing lyophilized enzyme powder, wherein the lyophilized enzyme powder includes recombinase capable of binding single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and Strand displacement DNA polymerase three enzymes, mix with a pipette gun to suspend all the particles in the tube, add 2.5 μl of 280 mM magnesium acetate solution, cover the tube cap, mix quickly, and react in a metal bath at 37°C for 4min, 4min After the end, take it out, turn it vigorously 8-10 times, mix well, ...

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Abstract

The invention discloses a primer and kit for using RPA for detecting procambarus clarki picornaviridae and a detection method. The primer for RPA detection is a primer pair of nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and is used for detecting or assisting in detecting procambarus clarki picornaviridae. The primer is used for detecting a to-be-detected sample of procambarus clarki,and if an RPA amplification product contains a fragment with the size of 151 bp, it is determined that to-be-detected procambarus clarki contains picornaviridae. The detection method comprises the steps of primer synthesis, RNA extraction in the to-be-detected sample, reverse transcription, RPA amplification, amplification product analysis and the like. The detection method has the advantages ofsimple operation and high stability, and a low-cost, fast and specific on-site diagnostic method is provided for effective detection and identification of procambarus clarki picornaviridae.

Description

technical field [0001] The invention belongs to the technical field of molecular detection of aquatic animal viruses, and relates to a method for detecting small RNA viruses of Procambarus clarkii using a recombinase-mediated isothermal amplification technology and special primers for detection. Background technique [0002] Procambarus clarkii (Procambarus clarkii), commonly known as crayfish, belongs to the phylum Arthropoda, the class Crustacea, and the genus Procambarus. Native to the southern United States and northern Mexico, it was introduced into China by Japan. Due to its high nutritional value and unique flavor, it has become an important freshwater economic aquatic animal and an important object of aquaculture in my country. [0003] However, in recent years, with the continuous expansion of the artificial breeding area of ​​Procambarus clarkii, diseases of Procambarus clarkii occur frequently, resulting in reduced yield and quality, and serious damage to farmers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 沈怀舜臧亚南胡亚成徐增洪水燕
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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