Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube
A real-time fluorescence quantitative and nucleic acid extraction technology, applied in the field of molecular biology, can solve the problems of unstable nucleic acid cleavage efficiency, nucleic acid loss, nucleic acid pollution, etc., and achieve the effect of optimized nucleic acid extraction effect, low cost, and complete protein removal
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[0075] Example 1: Real-time fluorescence quantitative PCR detection of HBVDNA
[0076] The reagents prepared according to the above-mentioned lysis solution solution 1) and washing solution solution 1) mixed with magnetic beads were used to make a clear clinical diagnosis and the quantitative value of HBVDNA after detection was 2×10 1 Perform the following steps with IU / ml hepatitis B patient serum:
[0077] (1) Aliquoting of PCR lysis solution: Aliquot the prepared lysis solution mixed with magnetic beads into a dedicated PCR tube according to 100μl per tube.
[0078] (2) Sampling: add 100 μl of serum to the above PCR tube containing the lysate mixed with magnetic beads, gently pipet and mix 5 times with a pipette, and let it stand at room temperature for 10 minutes.
[0079] (3) Aspirate and discard the liquid: place the PCR tube on the eight-row magnetic rack and let it stand for 2 minutes. Use a pipette to suck off the liquid on the opposite side of the magnetic beads. Be careful n...
Example Embodiment
[0087] Example 2: Real-time fluorescence quantitative PCR detection of HBVDNA
[0088] Reagents prepared according to the above-mentioned lysis solution solution 2) and washing solution solution 2) mixed with magnetic beads were used to make the clinical diagnosis clear and the HBVDNA quantitative value after detection was 2×10 1 Perform the following steps with IU / ml hepatitis B patient serum:
[0089] (1) Aliquoting of PCR lysis solution: Aliquot the prepared lysis solution mixed with magnetic beads into a dedicated PCR tube according to 100μl per tube.
[0090] (2) Adding samples: Take 100 μl of serum and add it to the above PCR tube containing the lysate mixed with magnetic beads, gently pipet and mix 5 times with a pipette, and let it stand at room temperature for 5 minutes.
[0091] (3) Aspirate and discard the liquid: place the PCR tube on the eight-row magnetic rack and let it stand for 2 minutes. Use a pipette to suck off the liquid on the opposite side of the magnetic beads. ...
Example Embodiment
[0099] Example 3: Sensitivity test of the real-time fluorescent quantitative PCR method of the present invention
[0100] Use HBVDNA-negative serum samples as diluents, the clinical diagnosis is clear and the quantitative value of HBVDNA detected is 2×10 9 The IU / ml serum of hepatitis B patients was diluted sequentially, and the HBVDNA concentrations were 5IU / ml, 10IU / ml, 20IU / ml, 2×10 2 IU / ml, 2×10 3 IU / ml, 2×10 4 IU / ml, 2×10 5 IU / ml, 2×10 6 IU / ml, 2×10 7 IU / ml, 2×10 8 IU / ml, 2×10 9 IU / ml sample. Using the same method as in Example 1, the above diluted samples were subjected to real-time fluorescent quantitative PCR detection.
[0101] The experimental results are as image 3 As shown, the curve from right to left thus represents the concentration of 5IU / ml, 10IU / ml, 20IU / ml, 2×10 2 IU / ml, 2×10 3 IU / ml, 2×10 4 IU / ml, 2×10 5 IU / ml, 2×10 6 IU / ml, 2×10 7 IU / ml, 2×10 8 IU / ml, 2×10 9 IU / ml sample. The results show that the present invention can detect the concentration range of 5IU / ml~...
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