One-step real-time fluorescent RT-PCR reaction buffer and reaction system and PCR method thereof
A technology of RT-PCR and reaction buffer, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., to achieve the effect of ensuring uniformity, high sensitivity, and strong temperature compatibility
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[0064] Preparation of template: Take 200 μL of H5N6 inactivated HI antigen purchased from Harbin Veterinary Research Institute, and extract viral RNA in an automatic nucleic acid extraction workstation according to the operation manual of the MagMAX nucleic acid extraction kit. Extract the template from 10 0 Serial 10-fold serial dilutions to 10 -10 , as a reaction template.
[0065] Hydrolysis probe H5-P sequence:
[0066] 5'-FAM-GGGTGATTATTTATTATTGTTGTTGTTGGGYTGGT-3'-BHQ1 (SEQ ID NO: 1), with a fluorescent group 5'-FAM (5'-carboxyfluorescein) at its 5' end and a quencher group at its 3' end BHQ1.
[0067]Upstream primer H5-F sequence: 5'-AAATTATTCTGAGGGTGATTATTTATTATTGT-3' (SEQ IDNO: 2)
[0068] Downstream primer H5-R sequence: 5'-ATCGGTAGTAAGCCTGCTAATAGGA-3'(SEQ IDNO:3)
[0069] 2) Reagent
[0070] Tris-HCl buffer (Tris-HCl) at a concentration of 1M, potassium chloride (KCl) at a concentration of 1M, magnesium chloride (MgCl) at a concentration of 1M 2 ), a concentra...
Example Embodiment
[0071] Example 1
[0072] 1) Configure one-step real-time fluorescent RT-PCR reaction buffer; mix Tris-HCl, KCl solution, MgCl 2 solution, (NH 4 ) 2 SO 4 Solution, DMSO, dATP, dGTP, dCTP, dTTP, Glycerol, BSA, Tween 20, NaN 3 Add the solution, Roxreference dye, and deionized water into the reaction container according to the concentration and ratio in the following table, and configure 100mL of one-step real-time fluorescent RT-PCR reaction buffer.
[0073] Table 1: Raw material concentration and proportioning in embodiment 1
[0074] Element
[0075] Where (2×) means that the concentration is doubled
[0076] 2) The reaction system formed by the one-step real-time fluorescent RT-PCR reaction buffer prepared by the present invention is as follows:
[0077] 25μL fluorescent RT-PCR reaction system:
[0078]
[0079] 3) The fluorescent RT-PCR reaction system was placed in a fluorescent quantitative PCR instrument for reaction, and the reaction was controlled at...
Example Embodiment
[0081] Example 2
[0082] 1) Configure one-step real-time fluorescent RT-PCR reaction buffer; mix Tris-HCl, KCl solution, MgCl 2 solution, (NH 4 ) 2 SO 4 Solution, DMSO, dATP, dGTP, dCTP, dTTP, Glycerol, BSA, Tween 20, NaN 3 Add the solution, Roxreference dye, and deionized water into the reaction container according to the concentration and ratio in the following table, and configure 100mL of one-step real-time fluorescent RT-PCR reaction buffer.
[0083] Table 2: Raw material concentration and proportioning in embodiment 2
[0084] Element
[0085] Where (2×) means that the concentration is doubled
[0086] 2) The reaction system formed by the one-step real-time fluorescent RT-PCR reaction buffer prepared by the present invention is as follows:
[0087] 25μL fluorescent RT-PCR reaction system:
[0088] 2×RT-PCR Buffer
[0089] 3) The fluorescent RT-PCR reaction system was placed in a fluorescent quantitative PCR instrument for reaction, and the den...
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