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Primers for cat coronavirus gene amplification and genotyping method

A feline coronavirus, genotyping technology, applied in the biological field, can solve the problems of difficult diagnosis, high incidence and difficult prevention, etc.

Pending Publication Date: 2020-08-11
LUDONG UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] FIP has the characteristics of high incidence, difficulty in diagnosis, difficulty in prevention, and almost irreversible death in a short period of time. At present, there is no effective long-term treatment plan. It is very important to establish and promote a fast and accurate diagnosis plan
However, affected by many factors, the reliability and practical operability of the existing FIP diagnostic methods are poor, so the market urgently needs a fast, simple and accurate new product or new technology for FIP diagnosis

Method used

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  • Primers for cat coronavirus gene amplification and genotyping method
  • Primers for cat coronavirus gene amplification and genotyping method
  • Primers for cat coronavirus gene amplification and genotyping method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 is established for feline coronavirus HRM detection and typing method

[0053] Step 1: Prepare Positive Control Standard

[0054] Entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize 3 fragments of the S sequence of the spike protein coding gene of the reference strain C1Je of feline coronavirus (GenBank accession number: 23400th to 23705th sequence in the genome of DQ848678), and named them respectively Fragment 1 , Fragment 2, Fragment 3, the 3 fragments respectively contain the 3 genotypes of this site of feline coronavirus, the sequence of Fragment 1 includes the genotype at the 23531st position of the genome, and the sequence of Fragment 2 includes the 23531st position of the genome as The genotype of C, the sequence of fragment 3 includes the genotype of A at position 23531 of the genome. Kpn I and BamH I restriction enzyme sites were introduced at the 5'-end and 3'-end of each synthetic S gene fragment, respectively, and named 23531T, 2...

Embodiment 2

[0084] Verify the sensitivity of the detection results of the real-time quantitative amplification of feline coronavirus using the primers described in Example 1 of the present invention. Include the following steps:

[0085] Dilute the RNA template: take the RNA prepared by in vitro transcription as the template (i.e. the three positive control standards prepared in Step 1 of Example 1), and after quantification, dilute the RNA copy number concentration to 10 with sterile enzyme-free deionized water. 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 5copies / μL and 10 0 copy / μL was used as the sample to be tested, and the negative control was used as 0 copy / μL template RNA.

[0086] Establishment of detection system

[0087] Set up the reaction system in 0.2mL PCR 8-strip tube, as follows:

[0088]

[0089] After the reaction system is prepared, gently mix the liquid in the micro reaction tube. Perform the following reaction:

[0090]

[0091] The reac...

Embodiment 3

[0094] Application of the HRM detection method using the primers described in Example 1 of the present invention in the clinical detection of feline coronavirus pets.

[0095] From August 2018 to November 2018, 42 samples of suspected FIP disease materials including plasma, serum and ascites were obtained from many pet hospitals in Changchun, Shenyang and Yantai. Detected by the method described in Example 1 of the invention. Among them, 6 samples had no obvious amplification curve, lower than the lower limit of detection, and the result was negative, 1 sample had a Ct value of 39, and the result was doubtful, and 37 samples had a Ct value ≤ 37, which was determined to be positive for feline coronavirus, which was the total sample size Among the 37 positive samples, 22 genotypes corresponding to the 23531th HRM amplification curve were T; 4 were identified as C, and 11 were identified as A in total. Based on this, it is inferred that there are 26 samples corresponding to the ...

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Abstract

The invention relates to a cat coronavirus gene amplification primer and a genotyping method. The primer is a cat coronavirus S gene primer, and a high-resolution melting curve (HRM) identification method of an SNP site (g.23531A>Y) on a cat coronavirus fibrin gene S can be established by applying the primer. A detection method established by applying the primer can be used for rapidly detecting and identifying the genotype of the 23531st site of the cat coronavirus genome from a pet clinical sample; and thus, and then the systemic infectivity phenotype of the corresponding strain can be deduced, so that the primer can be used as a basis for screening pathogenicity of the cat coronavirus, and can also be used as an important reference for clinical diagnosis of infectious peritonitis of cats.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer for feline coronavirus gene amplification and a detection and typing method. Background technique [0002] Feline coronavirus (FCoV) belongs to the Coronaviridae family and is a member of the genus Coronaviridae. According to the differences in clinical symptoms of infected cats, people are used to dividing FcoV into feline enteric coronavirus (FECV) and feline infectious peritonitis virus (Feline infectious peritonitis virus, FIPV). Among them, feline intestinal infection caused by FECV may cause dehydration, but the clinical symptoms are mild and the fatality rate is low. Feline infectious peritonitis (FIP) is an immune-mediated chronic and progressive infectious disease, characterized by peritonitis, pyogenic granulomatous vasculitis, massive ascites accumulation and high lethality. The disease mainly infects young cats under two years old, especially purebred cats are ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/11C12R1/93
CPCC12Q1/701C12Q2600/156C12Q2600/166
Inventor 朱洪伟王化磊黄清荣金宏丽张兴晓张建龙
Owner LUDONG UNIVERSITY
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