Fluorescence quantitative PCR reagent kit and detection method for human respiratory syncytial virus
A technology for syncytial virus and fluorescence quantification, which is applied in biochemical equipment and methods, microbe determination/inspection, biological testing, etc., to achieve the effects of improved sensitivity, strong specificity, and reliable results
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Embodiment 1
[0031] Example 1: Configuration of Human Respiratory Syncytial Virus Fluorescent Quantitative PCR Detection Kit
[0032] (1) RNA extract, M-MLV reverse transcriptase (200U / uL), dNTPs (10mM), HotStart TaqDNA polymerase (2.5U / uL), RNase inhibitor (40U / uL), MgCl2 (25mM).
[0033] (2) M-MLV 5×Buffer composition:
[0034] 50mM Tris-HCl (PH8.3, 25°C), 3mM MgCl 2 , 75mM KCl, 10mM DDT;
[0035] (3) Composition of fluorescent PCR 10×Buffer:
[0036] 200mM KCl, 200mM Tris-HCl (PH8.4, 25°C)
[0037] (4) Reverse transcription reaction solution: primer P12uL (10umol / L), M-MLV 5×buffer 4uL, dNTPs3uL (10mmol / L), sterile double distilled water 9uL.
[0038] (5) Fluorescence quantitative reaction solution:
[0039] PCR 10×buffer 2uL, P1, P2 each 0.5uL (10umol / L), fluorescent probe FP 1uL, (1umol / L), MgCl 2 2uL (25mmol / L), dNTPs 0.5uL (10mmol / L), sterile double distilled water 8uL.
Embodiment 2
[0040] Example 2: Detection of Respiratory Syncytial Virus with Fluorescent Quantitative PCR Detection Kit
[0041] (1) Take 300 μl of centrifuged cough sputum extract, nasal swab extract or throat swab extract, add 900 μL RNA extract, then add 200 μL chloroform, shake for 15 seconds, and incubate at room temperature for 10 minutes. Centrifuge at 12000g for 10min at 4°C, and the RNA is located in the upper layer. Replace the upper layer with a new 1.5ml EP tube, add 500ul isopropanol, incubate at -20°C for 10min, and then centrifuge at 12000g for 10min at room temperature. Discard the supernatant, add 1ml of 75% ethanol to wash the RNA pellet, shake and mix, and centrifuge at 12000g for 3min at 4°C. Discard the supernatant and dry at 37°C for 5 min. The precipitate was dissolved with 18uL reverse transcription reaction solution to obtain viral RNA.
[0042] (2) Add 1uL of RNase inhibitor and 1uL of M-MLV reverse transcriptase. 42°C for 60 minutes, 95°C for 5 minutes to ina...
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