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RT-RPA primer pair, probe, kit and detection method for detecting cat coronavirus

A technology of RT-RPA and feline coronavirus, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to achieve low temperature and machine requirements, high sensitivity, and short time-consuming effects

Active Publication Date: 2020-07-31
GUANGDONG LAB ANIMALS MONITORING INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the detection of Fcov using real-time fluorescent RPA at home and abroad.

Method used

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  • RT-RPA primer pair, probe, kit and detection method for detecting cat coronavirus
  • RT-RPA primer pair, probe, kit and detection method for detecting cat coronavirus
  • RT-RPA primer pair, probe, kit and detection method for detecting cat coronavirus

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1. is used for detecting the design and screening of the RT-RPA primer pair of feline coronavirus, probe

[0037] 1. Virus Extraction

[0038] RNA was extracted from ascites samples of cats according to the instructions of the RNA extraction kit from TIANGEN Company, and stored at -80°C for later use.

[0039] 2. Design of RT-RPA primers and probes

[0040] According to the recommendation of the TwistDX design guide, the Fcov sequence (accession number: KY566209.1) was downloaded from NCBI and RNAstar was used to design RT-RPA-specific primers and probes based on the highly conserved MP gene sequence. The designed primers range in length from 30 to 35 bases, and the probes range in length from 48 to 52 bases. The RPA probe should have four groups, including a fluorophore, a quencher, tetrahydrofuran (THF) and a 3-terminal blocker (C3spacer). All primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd. after the design was completed. ...

Embodiment 2

[0045] Embodiment 2. is used to detect the establishment of the RT-RPA method of feline coronavirus

[0046] Use the fluorescent RT-RPA kit of Hangzhou Zongce Biotechnology Co., Ltd. to carry out the RPA reaction with a 50uL reaction system. The optimal reaction system and conditions are: forward primer (10um) 2ul, reverse primer (10um) 2ul, RNA template 2ul, ABuffer 40.9ul, B Buffer 2.5ul, probe (10um) 0.6ul. The RT-RPA reaction was amplified at 39°C for 30 minutes in a real-time constant temperature fluorescence detector (Guangzhou Double Helix Gene Technology Co., Ltd.). Select a positive control and a negative control (both identified by qPCR) respectively, and carry out fluorescent RT-RPA identification, and the identification results are consistent with qPCR (see figure 2 ).

Embodiment 3

[0047] Embodiment 3, the present invention detects the RT-RPA method specificity test and sensitivity test of feline coronavirus

[0048] 3.1 Method

[0049] Specificity test: The specificity of the constructed real-time RPA method was determined by detecting nucleic acids containing Fcov-positive nucleic acids as well as nucleic acids from other pathogens. Nucleic acids from other pathogens include canine coronavirus (CCV), canine parvovirus (CPV), canine distemper (CDV), canine adenovirus (CAV), feline herpesvirus (FHV), feline parvovirus (FPV), cat cup Coronavirus (FCV). Sensitivity test: using plasmid diluted standards, with ddH 2 O was diluted to 7 concentrations at a ratio of 10 times, and the template copy numbers after dilution were 1*10 6 copies / μL to 1 copies / μL.

[0050] 3.2 Results

[0051] The specificity of the constructed real-time RPA method was determined by detecting nucleic acids containing Fcov-positive nucleic acids as well as nucleic acids from other...

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Abstract

The invention provides an RT-RPA primer pair, a probe, a kit and a detection method for detecting cat coronavirus, and belongs to the technical field of molecular biological detection. Aiming at an MPgene sequence of a cat coronavirus feline infectious peritonitis viruses (Fcov), a specific primer pair and a probe are designed, and a real-time fluorescence RPA detection method is established through repeated tests. The method disclosed by the invention is strong in amplification specificity and high in sensitivity, and can be used for detecting two viruses of FCoV-I and FCoV-II serotypes which both can cause feline infectious peritonitis (FIP) in one detection test, so that the method is more efficient. Technical reference is provided for rapid diagnosis and prevention and control of Fcov.

Description

technical field [0001] The invention relates to an RT-RPA primer pair, a probe, a kit and a detection method for detecting feline coronavirus, and belongs to the technical field of molecular biology detection. Background technique [0002] Feline coronavirus (FCoV) is a non-segmented, single-stranded positive-sense RNA virus belonging to the order Nidovirales, the family Coronaviridae, and the genus Alphacoronavirus. FCoV has two biotypes, Feline enteric coronavirus (FECV) and Feline infectious peritonitis viruses (FIPV). Although most cats infected with FCoV develop mild enteritis or are asymptomatic, 12% of cats develop feline infectious peritonitis (FIP), which is fatal. Based on the virus neutralizing antibody response and the difference in the amino acid sequence of the S gene, FCoV is divided into two serotypes, FCoV-I and FCoV-II, both of which can cause FIP. [0003] The existing experimental methods for detecting FCoV are mainly PCR methods, including ordinary PCR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/107C12Q2563/107C12Q2561/113Y02A50/30
Inventor 肖丽丛锋黄韧朱才毅
Owner GUANGDONG LAB ANIMALS MONITORING INST
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