H3 avian influenza virus two-temperature type RT-PCR detection kit and primer pair thereof
A technology of RT-PCR and avian influenza virus, applied in the field of H3 subtype avian influenza virus two-temperature RT-PCR detection kit and its primer pair, can solve the problems of only mild symptoms and low pathogenicity
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Embodiment 1
[0033] Embodiment 1, design and synthesis of primers
[0034] According to the conserved sequence of H3 subtype AIV HA gene in GenBank, DNAStar software was used for multiple sequence alignment, and primer5.0 was used to design primers in the conserved region. Primers were synthesized by Invitrogen. (Table 1).
[0035] Table 1 Primer information
[0036]
Embodiment 2
[0037] Embodiment 2, two-temperature RT-PCR detection
[0038] 1. Extraction of nucleic acid and reverse transcription of RNA
[0039]Refer to the instructions of the DNA / RNA extraction kit to extract the DNA of infectious laryngotracheitis and Mycoplasma gallisepticum, and at the same time conduct H1, H3, H4, H6, H7, H9, poultry respiratory infectious diseases, infectious bronchitis and Newcastle disease virus Extract, and reverse transcribe RNA into cDNA according to the reverse transcription instructions, as follows: Use 50 μL system, add 10 μL 5× reverse transcription buffer, 10 mmol / L dNTP4 μL, 5U / μL MV1 μL, 20U / μL in sequence in EP tube 1 μL of RNase inhibitor, 2 μL of influenza universal primer (sequence 5′-AGCAAAAGCAGG-3′, SEQ.ID.No.3), 32 μL of total RNA template to be tested, and the prepared cDNA Store at -30°C for later use.
[0040] 2. Establishment of two-temperature RT-PCR amplification system
[0041] The total reaction system is 25 μL, including 12.5 μL of ...
Embodiment 3
[0048] Embodiment 3, the assembly of detection kit
[0049] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.
[0050] The kit includes a primer pair, PCR buffer and water; the primer pair includes primers 1 and 2, the concentration of which is 25 μmol / L (the final concentration in the PCR reaction system is 0.5 μmol / L); the PCR buffer is 2× Taq PCR Mix.
[0051] Other test solutions in PCR detection can be configured on site or directly use commercial reagents.
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