HRM primer pair, kit and method for rapidly identifying feline enteric coronavirus and feline infectious peritonitis viruses

A technology of peritonitis virus and coronavirus, applied in the field of molecular biology detection

Pending Publication Date: 2020-08-11
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Currently, there is no report on the iden

Method used

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  • HRM primer pair, kit and method for rapidly identifying feline enteric coronavirus and feline infectious peritonitis viruses
  • HRM primer pair, kit and method for rapidly identifying feline enteric coronavirus and feline infectious peritonitis viruses
  • HRM primer pair, kit and method for rapidly identifying feline enteric coronavirus and feline infectious peritonitis viruses

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Experimental program
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Embodiment 1

[0036] Embodiment 1. is used to distinguish the design of the HRM primer of feline enteric coronavirus and feline infectious peritonitis virus

[0037] 1.1 Virus isolation and cultivation and nucleic acid extraction

[0038] FIPV was isolated and cultured in our laboratory, and FECV nucleic acid was extracted from clinical samples. Nucleic acid RNA was extracted from cat ascites and intestinal feces samples according to the instructions of the RNA extraction kit from TIANGEN.

[0039] 1.2 Design of HRM primers

[0040] The full-length sequences of FECV and FIPV were downloaded from NCBI (Genebank accession numbers are KY566209 and KC461237.1, respectively) for comparison. Attempts to design primers on the commonly used conserved gene S or N gene were unsuccessful. Finally, a pair of primers were designed on a relatively conserved sequence on the 7b gene (between 29 000 bp and 29 500 bp) (primer sequences are shown in Table 1). The pair of primers can specifically amplify a...

Embodiment 2

[0045] Example 2. Establishment of the HRM Response for Distinguishing Feline Enteric Coronavirus and Feline Infectious Peritonitis Virus

[0046] Using the extracted nucleic acid as a template, use the upstream and downstream primers in Table 1 to amplify. A total of 25 μl of RT-PCR reaction system: RNA 1 μl, PrimerScript 1step Enzyme 1 μl, 2×step Buffer 12.5 μl, RNase Free ddH 2 O 5.5 μl, upstream primer 1 μl, downstream primer 1 μl, fluorescent dye LC GREEN 1 μl. The working concentration of upstream and downstream primers was 10 μmol / L.

[0047] The RT-PCR reaction program was 50°C for 30 minutes and 95°C for 2 minutes. Pre-denaturation at 95°C for 30s; 35 cycles at 95°C for 10s, 55°C for 20s, 72°C for 30s, 72°C for 5min, 4°C for 30min. In the HRM analysis, it is 75°C-88°C, with a heating rate of 0.1°C / step, collecting fluorescence every 0.05s for melting curve analysis.

[0048] The results show that the present invention has successfully established a HRM method for si...

Embodiment 3

[0049] Embodiment 3, the present invention distinguishes the HRM method specificity test of feline enteric coronavirus and feline infectious peritonitis virus

[0050] In order to analyze whether the HRM reaction has detection specificity in other canine and cat pathogens on the basis of being able to distinguish FECV and FIPV, the nucleic acid of the following samples was used as a template for HRM reaction: canine coronavirus (CCV), canine parvovirus (CPV ), canine distemper (CDV), canine adenovirus (CAV), feline herpesvirus (FHV), feline parvovirus (FPV), feline calicivirus (FCV), a negative control (ddH 2 0), one FECV positive sample, one FIPV positive sample.

[0051] The results showed that all samples were negative except for FECV and FIPV (see figure 2 ). It proved that the method has good specificity and no cross-reaction with other pathogens.

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Abstract

The invention provides an HRM primer pair, kit and method for rapidly identifying feline enteric coronavirus and feline infectious peritonitis viruses, and belongs to the technical field of molecularbiological detection. The high-resolution melting (HRM) primer pair disclosed by the invention is designed according to a sequence between 29000bp and 29500bp of a full-length gene of feline coronavirus (FCOV). The feline enteric coronavirus (FECV) and the feline infectious peritonitis viruses (FIPV) can be identified and distinguished at the same time through melting curves with different shapesby one-time reaction, and technical support is provided for early diagnosis and prevention and control of diseases.

Description

technical field [0001] The invention relates to a pair of HRM primers, a kit and a method for quickly identifying feline intestinal coronavirus and feline infectious peritonitis virus, 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. [0003] The existing experimental methods for detecting FECV and FCoV are mainly PCR methods, including ordinary PCR and fluorescent quantitative PCR, as well as indirect immunofluorescence and ELISA methods. PCR technology is widely used due to its high detect...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2527/107C12Q2521/107
Inventor 丛锋肖丽黄碧洪
Owner GUANGDONG LAB ANIMALS MONITORING INST
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