Real-time fluorescent RT-PCR detection method for MARV (Marburg virus)
A RT-PCR and real-time fluorescence technology, applied in the biological field, can solve problems such as increased risk, and achieve the effects of improved sensitivity, strong detection specificity, and good amplification effect
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Embodiment 1
[0027] A set of real-time fluorescent RT-PCR primers and probes for detection of monkey Marburg virus, including upstream primer MbV-F, downstream primer MbV-R and TaqMan probe MbV-P, the length of the amplified target fragment is 163bp, such as figure 1 As shown, the nucleotide sequences of each primer are as follows:
[0028] Upstream primer MbV-F: 5'- TTG GTG CGG ACC TCC AAA GT -3';
[0029] Downstream primer MbV-R: 5'- GCG CAA TTG CTG AGA GCT GA -3';
[0030] TaqMan probe MbV-P: 5'- FAM-AGC AGG CGT TGA GCA ACC TAG CCC GA- 3'.
[0031] The 5' and 3' ends of the probe were modified with fluorescent groups FAM and BHQ1, respectively.
Embodiment 2
[0033]Optimization of annealing temperature for real-time fluorescent RT-PCR detection method of monkey Marburg virus:
[0034] (1) Real-time fluorescent RT-PCR reaction system: 10×RT-PCR buffer 2 μL, 25 mmol / L MgCl 2 2 μL, 2.5 mmol / L dNTPs 1.5 μL, 10 μmol / L upstream and downstream primers 0.5 μL each, 10 μmol / L probe 0.5 μL, 5 U / μL DNA polymerase 0.5 μL, 5 U / μL reverse transcriptase 0.5 μL, 40 U / μL RNase inhibitor 0.5 μL, sample RNA to be tested 4 μL, then add 7.5 μL of DEPC water to make the total reaction volume 20.0 μL;
[0035] (2) Real-time fluorescence RT-PCR optimized reaction program: reverse transcription at 50°C for 30 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C-65°C (gradient temperature) for 30 s The temperature setting was repeated 3 times, a total of 40 cycles;
[0036] (3) Selection of annealing temperature: the optimization results of annealing temperature are as follows: figure 2 As shown, when the annealing ...
Embodiment 3
[0038] Utilize the detection method that the simian Marburg virus real-time fluorescent RT-PCR primer and probe optimization provided by the invention comprise the following steps:
[0039] (1) Real-time fluorescent RT-PCR reaction system configuration: 10×RT-PCR buffer 2 μL, 25 mmol / L MgCl 2 2 μL, 2.5 mmol / L dNTPs 1.5 μL, 10 μmol / L upstream and downstream primers 0.5 μL each, 10 μmol / L probe 0.5 μL, 5 U / μL DNA polymerase 0.5 μL, 5 U / μL reverse transcriptase 0.5 μL, 40 U / μL RNase inhibitor 0.5 μL, sample RNA to be tested 4 μL, then add 7.5 μL of DEPC water to make the total reaction volume 20.0 μL;
[0040] (2) Real-time fluorescent RT-PCR reaction program: reverse transcription at 50°C for 30 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 61°C for 30 s, a total of 40 cycles, and single-point fluorescence detection at 61°C ;
[0041] (3) Judgment of results: the negative control has no Ct value and no amplification curve, and the positi...
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