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Primer set, reagent kit and method for detecting canine distemper viruses, canine parvoviruses and canine coronaviruses

A technology for canine parvovirus and canine distemper virus, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection. , good specificity and good accuracy

Inactive Publication Date: 2018-08-07
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the defects and deficiencies that traditional methods cannot detect canine distemper virus (Canine distemper virus, CDV), canine parvovirus (Canine parvovirus, CPV) and canine coronavirus (Canine coronavirus, CCV) at one time, and provides a method based on 3-fold PCR method designed for the detection of canine distemper virus (Canine distempervirus, CDV), canine parvovirus (Canine parvovirus, CPV) and canine coronavirus (Canine coronavirus, CCV) detection primer set and the kit based on the primer set and The corresponding detection method can carry out early rapid, sensitive and accurate identification of single or mixed infection of canine distemper virus (Canine distemper virus, CDV), canine parvovirus (Canine parvovirus, CPV) and canine coronavirus (Canine coronavirus, CCV)

Method used

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  • Primer set, reagent kit and method for detecting canine distemper viruses, canine parvoviruses and canine coronaviruses
  • Primer set, reagent kit and method for detecting canine distemper viruses, canine parvoviruses and canine coronaviruses
  • Primer set, reagent kit and method for detecting canine distemper viruses, canine parvoviruses and canine coronaviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the primer set described in the present invention

[0052] According to the related sequence information of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) published on GenBank (Table 1), different canine distemper virus (CDV), canine parvovirus (CPV) were analyzed and the conserved region of canine coronavirus (CCV), on the basis of the analysis of the virus conserved region, primers were designed and synthesized by Invitrogen. After a large number of screening tests, primer pairs that can simultaneously amplify target fragments in the same system with excellent specificity and high sensitivity were obtained (Table 2).

[0053] Table 1 CDV, CPV and CCV sequences referenced in this test

[0054] Table 1 CDV, CPV and CCV sequences used in this study

[0055]

[0056] Table 2 Primers used in this test

[0057]

[0058] Thus obtain the primer set of the present invention, including three pairs corresponding to canin...

Embodiment 2

[0059] Embodiment 2, kit according to the present invention

[0060] Based on the primer set obtained in Example 1, the triple PCR kit for detecting CDV / CPV / CCV described in the present invention was assembled. The kit includes: a primer set including three pairs of upstream and downstream primer pairs respectively corresponding to CDV / CPV / CCV and having the nucleotide sequences shown in Table 2.

[0061] The kit also includes: reagents required for PCR reaction and / or electrophoresis. Specifically, the conventional reagents for the PCR reaction include: common commercially available PCR kits, PCR reaction Mix, etc., such as 2×Es Taq Master Mix; the reagents required for the electrophoresis include electrophoresis buffer, Agarose, ethidium bromide (EB), etc.

Embodiment 3

[0062] Embodiment 3, performance verification of the primer set / kit of the present invention

[0063] 1 method

[0064] 1.1 Preparation of standard products

[0065] The concentration of positive plasmids pEASY-D, pEASY-P and pEASY-C containing CDV, CPV and CCV genome fragments was detected with a nucleic acid analyzer. According to the concentration determination results, the positive plasmid was treated with ddH 2 Dilute with O, and dilute the concentrations of the three plasmids to 20ng / μL, and then mix them in equal amounts to obtain the plasmid standard. 1.2 Optimization of reaction conditions

[0066] The PCR reaction system established in the present invention is 25 μL, in which 12.5 μL of 2×Es Taq Master Mix, 3 μL of pEASY-D, pEASY-P and pEASY-C positive mixed plasmid templates are added, and the initial concentration of primers is 10 μmol / L. Adjust the volume between 0.5μL-1.5μL to determine the most suitable primer volume. On the basis of determining the optimal...

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Abstract

The invention provides a primer set, a reagent kit and a method for detecting canine distemper viruses (CDV), canine parvoviruses (CPV) and canine coronaviruses (CCV), and belongs to the technical field of virus detection. The primer set comprises primers with nucleotide sequences shown as SEQ ID No.1-6. The primers correspond to three pairs of upstream and downstream primer pairs for the canine distemper viruses (CDV), the canine parvoviruses (CPV) and the canine coronaviruses (CCV). The reagent kit comprises the primer set. The method is based on the primer set and the reagent kit and is used for detecting the canine distemper viruses, the canine parvoviruses and the canine coronaviruses. The primer set, the reagent kit and the method have the advantages that the canine distemper viruses, the canine parvoviruses and the canine coronaviruses can be detected by the aid of the primer set, the reagent kit and / or the method, the primer set, the reagent kit and the method are good in specificity and accuracy and high in sensitivity and efficiency and are simple and speedy, reliable technical means can be provided to clinical detection, and the like.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a primer set, a kit and a PCR method for detecting canine distemper virus, canine parvovirus and canine coronavirus. Background technique [0002] Canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) are the main pathogens causing canine disease, and these three viruses are clinically fatal after single or mixed infection The rate can reach 50% to 100%, causing serious economic losses to the dog industry. Canine distemper caused by canine distemper virus mainly includes respiratory type, digestive type, neurological type and mixed type. Canine parvovirus mainly causes acute hemorrhagic gastroenteritis in dogs and acute myocarditis in puppies; canine coronavirus usually causes mild in dogs. A severe, self-limiting intestinal disorder characterized by sudden onset of vomiting and foul-smelling diarrhea in affected dogs. [0003] T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2537/143Y02A50/30
Inventor 杨兵周宏专苏霞常彦嫣
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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