Fluorescence quantitative RT-PCR kit for detecting yellow fever viruses, detection method and application thereof
A technology of yellow fever virus and RT-PCR, which is applied in the field of fluorescent quantitative RT-PCR kits for detecting yellow fever virus, and can solve the problems of no application kit and absence of nucleic acid, etc.
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Embodiment 1
[0038] Embodiment 1: the preparation of positive control RNA template
[0039] According to the sequence of yellow fever virus (GenBank sequence number: NC_002031), amplify a DNA fragment with a size of 606bp containing the fragment sequence detected by fluorescent RT-PCR, clone it on the pGEM-T easy vector, and purify the plasmid with a plasmid DNA extraction kit DNA, sequenced to confirm orientation of insert. Use universal primers paired with the vector to amplify the T7 promoter and the inserted yellow fever sequence, then use MAXIscript T7 Kit for in vitro transcription to generate target RNA, and then use TURBO DNase for DNase treatment to remove untranscribed PCR amplification The template is finally precipitated with ethanol, and the RNA product is purified, which is the positive control RNA template, and stored in a -70°C ultra-low temperature refrigerator until use.
[0040] Take 5 μl of template RNA for in vitro transcription, dilute 150 times, and measure OD 260 ...
Embodiment 2
[0042] Example 2: Determination of the amplification program
[0043] The kit used for RT-PCR reaction is Ag-Path-ID TM One step RT-PCR Kit (product of Ambion, USA), amplification and detection were carried out on Roche Lightcycler1.5 real-time fluorescent quantitative PCR instrument.
[0044] According to the specific characteristics of the primers and probes, the reaction program on the instrument tested 3 temperatures and 2 times in the selection of annealing extension conditions: the reverse transcription reaction conditions were 45°C for 10 minutes, 10 minutes after hot start at 95°C, and then entered 45°C. Two-step PCR cycle: 95°C for 5 seconds → (58 / 60 / 62)°C (20 / 30) seconds; the sample volume of each reagent is 10 μl of 2×RT-PCR buffer in each reaction tube , 10 μM forward primer YFV-FP 1 μl, 10 μM reverse primer YFV-RP 1 μl, 5 μM probe YFV-probe 1 μl, 25×RT-PCR Enzyme Mix 0.8 μl, the amount of RNA is 5 μl, provided by the kit Water was added to the total reaction vo...
Embodiment 3
[0046] Embodiment 3: Determination of optimal primer concentration
[0047] Applying the above amplification procedure, the final concentration of the fixed probe is 250nM, and the concentration of the forward and reverse primers is selected among 250nM, 500nM and 750nM. Select the appropriate primer concentration according to the concentration listed in Table 2 plus the final concentration of downstream primers.
[0048] Table 2
[0049]
[0050] Such as figure 1 Shown is the graph of the experimental results of the determination of the optimal primer concentration. The primer concentration corresponding to the curve with the smallest Ct value and the obvious amplification curve is the optimal concentration, see figure 1 . The final concentration of YFV-FP was determined to be 500nM, and the final concentration of YFV-RP was 750nM.
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