Primer pair, probe, kit and detection method for detecting avian influenza virus h7 subtype
A technology of avian influenza virus and primer pair, applied in the field of primer pair detection of avian influenza virus H7 subtype, can solve the problems of high sensitivity and achieve the effects of high sensitivity, short time and good application prospects
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Embodiment 1
[0059] Example 1: Screening tests for specific primers and probes
[0060] 1. Design of primers and probes for H7 subtype of avian influenza virus
[0061] The inventors designed multiple pairs of real-time fluorescent RT-PCR (FQ-PCR) amplification primers and TaqManMGB probes for H7 subtype of avian influenza virus, which were synthesized by Shanghai Jikang Biotechnology Co., Ltd. The specific sequence is shown in Table 1, for example.
[0062] Table 1: Primers and probes for real-time fluorescent RT-PCR screening of avian influenza virus H7 subtypes
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[0064] 2. The reaction system and conditions of the real-time fluorescent RT-PCR method for H7 subtype of avian influenza virus
[0065] The reaction system of the H7 subtype real-time fluorescent RT-PCR method is shown in Table 2, and the reaction conditions are shown in Table 3.
[0066] Table 2: Reaction system of avian influenza virus H7 subtype real-time fluorescent RT-PCR method
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Embodiment 2
[0083] Example 2: Optimization of the Real-time Fluorescent RT-PCR Detection Method for Avian Influenza Virus H7 Subtype
[0084] 1. Design of primers and probes for avian influenza virus H7 subtype real-time fluorescent RT-PCR method
[0085] Refer to Example 1 for the primer and probe design of the H7 subtype real-time fluorescent RT-PCR method, and see Table 4 for the specific sequence.
[0086] 2. The reaction system and conditions of the real-time fluorescent RT-PCR method for H7 subtype of avian influenza virus
[0087] The optimization principle of the real-time fluorescent RT-PCR method for H7 subtype of avian influenza virus is: to obtain the maximum amplification efficiency and the minimum Ct value for the same sample by optimizing the following conditions.
[0088] (1) Determination of the optimal fluorescent primer concentration: the fluorescent primer concentration was screened between 300nM and 800nM.
[0089] (2) Determination of the optimal probe concentratio...
Embodiment 3
[0112] Embodiment 3: The composition of the real-time fluorescent RT-PCR detection kit of avian influenza virus H7 subtype
[0113] 1. RT-PCR reaction buffer: Each reaction includes a reaction buffer (containing MgCl 2 and dNTP) 12.5 μL, aliquoted into 1 mL serum tubes, 750 μL / tube, and stored at -20°C;
[0114] 2. Fluorescent probes and primers: Each reaction includes primers (AIV-H7-F1 and AIV-H7-R2) and probe AIV-H7-P1 at final concentrations of 600nM and 240nM, respectively, and stored at -20°C;
[0115] 3. Enzyme mixture: Each reaction includes high-efficiency reverse transcriptase and hot-start Taq enzyme, stored at -20°C;
[0116] 4. Sterile nuclease-free water: aliquot into 0.5mL serum tubes, 600μL / tube, and store at -20°C;
[0117] 5. Positive control: A fragment of about 200 bp in the HA region of the specific clone of avian influenza virus H7 subtype H7N2 was obtained by reverse transcription in vitro, with a Ct value <30, and stored at -20°C.
[0118] 6. Negativ...
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