A double eva Green real-time fluorescent quantitative PCR detection kit for detecting type I and type IV equine herpes virus and its application

A real-time fluorescence quantitative, equine herpes virus technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem of inability to achieve rapid detection, and achieve the effect of high positive detection rate

Inactive Publication Date: 2016-03-30
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Researchers at home and abroad have established various common PCR, SYBRGreen fluorescent quantitative PCR, and TaqMan fluorescent quantitative PCR methods to detect EHV-1 and EHV-4, but none of them can achieve the goal of fast detection and accurate and reliable results.

Method used

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  • A double eva Green real-time fluorescent quantitative PCR detection kit for detecting type I and type IV equine herpes virus and its application
  • A double eva Green real-time fluorescent quantitative PCR detection kit for detecting type I and type IV equine herpes virus and its application
  • A double eva Green real-time fluorescent quantitative PCR detection kit for detecting type I and type IV equine herpes virus and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 The establishment of double EvaGreen real-time fluorescent quantitative PCR detection method and its application in the detection of type I and type IV equine herpes virus

[0031] 1. Materials and methods

[0032] 1.1 Materials

[0033] 1.1.1 Virus reference strains Equine herpesvirus type I (EHV-1) virus strain (ATCC438 / 77), equine herpesvirus type IV (EHV-4) virus strain (ATCC405 / 76). RK13 cells were preserved by the Equine Disease Research Laboratory of the State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0034] 1.1.2 Clinical samples In 2013, nasal swabs of 11 horses with no characteristic clinical manifestations and 2 donkeys with no clinical manifestations were collected.

[0035] 1.1.3 Main reagents and instruments ViralDNAKit (Omega Company); 2XSsoFastTMEvaGreensupermix (Bio-Rad Company); fluorescent quantitative PCR eight-tube (Axygen Company). SratageneMx3005P fluo...

Embodiment 2

[0069] Embodiment 2 is used to detect the preparation of the dual EvaGreen real-time fluorescent quantitative PCR detection kit of type I and type IV equine herpes virus

[0070] 1. Primer design and synthesis

[0071] All published nucleotide sequences of EHV-1 and EHV-4 were downloaded from GenBank. Sequences were compared and analyzed using SeqMan and MagAlign; EHV-1 or EHV-4 type-specific primers were designed using Primer Premier 5.0 (Primer Biosoft international, palo Alto, CA, USA) software for the detection of EvaGreen fluorescent quantitative PCR.

[0072] The primer sequences used to detect equine herpesvirus type I are shown in SEQ ID NO.1 (upstream) and SEQ ID NO.2 (downstream), and the primer sequences used to detect equine herpesvirus type IV are shown in SEQ ID NO.3 (upstream) and SEQ ID NO. 4 (downstream).

[0073] 2. Preparation of double reaction solution for detecting type I and type IV equine herpes virus:

[0074] The double reaction solution for detect...

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Abstract

The invention discloses a dual Eva Green real-time fluorescence quantification PCR (Polymerase Chain Reaction) detection kit for detecting equine herpesviruses I and IV and application thereof. The detection kit is characterized in that Eva Green is taken as a fluorescent dye, and meanwhile, the detection kit further contains dual reaction liquid for detecting the equine herpesviruses I and IV. According to the application of the detection kit, an acquired positive clone plasmid and extracted virus genome DNA (deoxyribonucleic acid) are taken as templates to evaluate the sensibility, specificity and the like of dual Eva Green fluorescence quantification PCR utilizing the detection kit; a result shows that the detection kit has the characteristics of rapidness, reliability, high sensitivity and the like when being used for carrying out the dual Eva Green fluorescence quantification PCR on the equine herpesviruses I (EHV-1) and IV (EHV-4); furthermore, by utilizing the detection kit, the EHV-1 and the EHV-4 can be simultaneously detected in a single reaction, the positive rate of inapparent infection is high, and the early diagnosis and control of equine rhinopneumonitis can be realized.

Description

technical field [0001] The invention relates to a PCR detection kit for detecting type I and type IV equine herpes virus and its application, in particular to a dual EvaGreen real-time fluorescence quantitative PCR detection kit for detecting type I and type IV equine herpes virus The invention and an application thereof belong to the field of virus diagnosis and detection. Background technique [0002] Equinerhinopneumonitis (ER) is a highly contagious disease of equine animals caused by equine rhinopneumonia virus (ERV), characterized by fever and respiratory tract catarrh. The International Office of Epizootics (OIE) listed equine rhinopneumonia as a category B disease and was included in the list of legally notifiable diseases by the International Animal Health Code (1999). The Ministry of Agriculture of my country lists it as three types of animal diseases, and it is also listed as one of the diseases of entry-exit equine animals in the quarantine clauses signed by the...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143C12Q2563/107C12Q2561/113
Inventor王晓钧胡哲
OwnerHARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI