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HRM primer set for identifying CPV and FPV, kit containing primer set, and applications of kit

A kit and primer set technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., to achieve good sensitivity and specificity

Inactive Publication Date: 2018-08-03
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in addition to DNA sequencing, the only way to diagnose CPV and FPV typing is to use the hemagglutination inhibition test of monoclonal antibodies to distinguish

Method used

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  • HRM primer set for identifying CPV and FPV, kit containing primer set, and applications of kit
  • HRM primer set for identifying CPV and FPV, kit containing primer set, and applications of kit
  • HRM primer set for identifying CPV and FPV, kit containing primer set, and applications of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Primer Design

[0031] Download the whole genome sequences of canine parvovirus and feline parvovirus from GenBank, and find stable gene fragments with base differences through MEGA7 comparison analysis (reference strain: CPV Laika-1993, accession number: JN033694.1), design A pair of qPCR primers (F2 / R2) for distinguishing between CPV and FPV. A pair of primers (F1 / R1) were designed to amplify gene fragments including qPCR amplified fragments to construct CPV and FPV standard plasmids. The primer sequences are shown in Table 1.

[0032] Table 1 Primer Sequence

[0033]

Embodiment 2

[0034] The establishment of embodiment 2 high resolution melting curve (HRM) method

[0035] 1. Construction of standard plasmids

[0036] Take 200ul CPV virus stock solution and FPV virus stock solution respectively, and extract virus DNA. The operation steps were carried out according to the operating instructions of TAKARA's MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0. Use the DNA extracted by the kit as a template, use F1 / R1 (Table 1) as a primer, and refer to Taq TM (Ex Taq TM Version 2.0plus dye) (Takara, RR902A) operating instructions for PCR amplification, PCR system (50ul) as shown in Table 2.

[0037] Table 2 PCR system (50ul)

[0038]

[0039]

[0040] The pre-amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50°C for 1 min, extension at 72°C for 2 min; cycle 35 times; final extension at 72°C for 10 min.

[0041] Prepare a 3% gel with diluted buffer (1×TAE). Take 5ul of the P...

Embodiment 3

[0053] Embodiment 3 clinical application

[0054] Collect 80 clinical samples suspected of being infected with canine parvovirus or feline parvovirus from Beijing, Shenyang, Shijiazhuang and Changchun, conduct clinical testing according to the HRM method established in Example 2, and compare with tests such as virus isolation and DNA sequence determination Compare. The results of HRM analysis revealed that there were 42 CPV positive samples and 6 FPV positive samples. 45 samples were successfully sequenced, 39 samples were positive for CPV and 6 samples were FPV positive. The virus isolation test was carried out on 80 samples, and 32 strains of CPV and 4 strains of FPV were obtained. Compared with DNA sequencing, the coincidence rate, relative specificity and sensitivity of the HRM method were 96.25%, 91.43%, and 100% respectively; compared with the virus isolation test, the coincidence rate, relative specificity and sensitivity of the HRM method were 85% , 72.72%, 100%.

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Abstract

The present invention discloses an HRM primer set for identifying canine parvovirus (CPV) and feline parvovirus (FPV), a kit containing the primer set, and applications of the kit, wherein the primerset comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is represented by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is represented by SEQ ID NO.2. The present invention further provides an HRM analysis kit containing the primer set. According to the present invention, the experiment results prove that the kit has good sensitivity and good specificity in the detection of FPV and CPV, and has high coincidence, high relative specificity and high sensitivity compared to virus isolation, DNA sequencing and other tests; and thekit can provide the effective technical means for the prevention and control of diseases caused by canine parvovirus (CPV) and feline parvovirus (FPV).

Description

technical field [0001] The invention relates to a virus detection primer, a kit containing the primer and application thereof, in particular to an HRM primer for distinguishing FPV and CPV, a kit containing the primer and application thereof. The invention belongs to the technical field of virus detection and diagnosis. Background technique [0002] Parvoviruses (Parvoviridae) are single-stranded, linear DNA viruses with an average genome size of 5000 nt. According to the latest division of the International Committee on Taxonomy of Viruses in 2016, parvoviruses are divided into the parvovirus subfamily that can infect mammals and the densovirus subfamily that can infect insects. Canine parvovirus (Canine parvovirus, CPV) and feline parvovirus (Feline parvovirus, FPV) are members of the subfamily Parvoviridae and the genus Canine parvovirus. [0003] CPV and FPV are the main infectious pathogens of domestic dogs, cats and wild carnivorous animals, with a genome similarity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2527/107C12Q2561/113
Inventor 王建科程悦宁孙亚茹程世鹏林鹏易立仝明薇
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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