Multiplex fluorescence quantitative PCR detection kit for genotyping of avian influenza virus n subtype and its application
A multiplex fluorescence quantitative, avian influenza virus technology, applied in the determination/inspection of microorganisms, microorganism-based methods, microorganisms, etc., can solve the problems of poor specificity and low sensitivity
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Embodiment 1
[0066] Embodiment 1 Preparation of a nucleic acid typing detection kit for rapid detection of N subtype of avian influenza virus
[0067] The kit includes nucleic acid amplification reaction solution, enzyme mixed solution, avian influenza virus N subtype reaction solution A, avian influenza virus N subtype reaction solution B, avian influenza virus N subtype reaction solution C, positive control A, positive Control B, positive control C and blank control;
[0068] Wherein, the nucleic acid amplification reaction liquid includes Tris, potassium chloride, magnesium chloride, and nucleotide mixture;
[0069] Avian Influenza Virus N Subtype Reaction Solution A contains the following components:
[0070] Component (1): consists of six primers for detecting the N1-3 subtype of avian influenza virus; wherein, the base sequences of the six primers are SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, SEQ ID No.3, SEQ ID No. Shown in ID No.4, SEQ ID No.5 and SEQ ID No.6; The primer concentr...
Embodiment 2
[0087] Application of the kit of the present invention in the genotyping of N subtype of avian influenza virus of embodiment 2
[0088] 1. Extract sample RNA
[0089] 1.1 Take 200ul of clinical samples, add 400ul of Poly(A)-added Binding Buffer, mix thoroughly, transfer to a high-purification filter tube, centrifuge at 8000rmp for 15s, and discard the waste liquid in the collection tube.
[0090] 1.2 Add 500ul Inhibitor Removal Buffer to the filter tube, centrifuge at 8000rmp for 1min, and discard the waste liquid in the collection tube.
[0091] 1.3 Add 450ul Washing Buffer to the filter tube, centrifuge at 8000rmp for 1min, and discard the waste liquid in the collection tube.
[0092] 1.4 Repeat step 1.3, and then centrifuge at high speed for 10s. Be sure to remove the waste liquid in this step.
[0093] 1.5 Add 50ul Elution Buffer to the filter tube, let stand at room temperature for 2min, centrifuge at 8000rmp for 1min, and the solution obtained by centrifugation is the ...
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