Dual Eva Green real-time fluorescence quantification PCR (Polymerase Chain Reaction) detection kit for detecting equine herpesviruses I and IV and application thereof
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
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Embodiment 1
[0030] Example 1 The establishment of double Eva Green 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 Viral DNA Kit (Omega Company); 2X SsoFastTM EvaGreen supermix (Bio-Rad Company); fluorescent quantitative PCR eight-tube (Axygen Company). Sratagene Mx300...
Embodiment 2
[0069] Embodiment 2 is used to detect the preparation of the dual Eva Green 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 aligned and analyzed using SeqMan and MagAlign; EHV-1 or EHV-4 type-specific primers were designed using Primer Premier5.0 (Primer Biosoft international, palo Alto, CA, USA) software for Eva Green fluorescent quantitative PCR detection.
[0072] The primer sequence for detecting equine herpesvirus type I is shown in SEQ ID NO.1 (upstream) and SEQ ID NO.2 (downstream), and the primer sequence for detecting equine herpesvirus type IV is shown in SEQ ID NO.3 (upstream) and shown in 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 detecting ...
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