Fluorescent quantitative PCR primers and kit for detecting novel SADSCoV (swine acute diarrhea syndrome coronavirus)
A technology for acute diarrhea and fluorescence quantification, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., to achieve high accuracy, efficient identification, and good repeatability
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Embodiment 1
[0046] The preparation of embodiment 1 standard positive template
[0047] 1. Extraction of total virus RNA
[0048] According to the Invitrogen company TRIZOL LS Reagent RNA extraction kit instruction manual. Add 250 μL of the aliquoted supernatant of positive disease material grinding liquid (from a pig farm in Guangdong) and 750 μL TRIZOL to a 1.5 mLeppendorf tube, mix thoroughly, and place at room temperature for 10 min; add 200 μL of chloroform, shake vigorously for 15 sec, and stand at room temperature for 5 min. Centrifuge at 12000rpm at 4°C for 15min; transfer the supernatant to a new 1.5mL eppendorf tube, add 500μL of isoamyl alcohol, mix well, place at room temperature for 10min, centrifuge at 12000rpm at 4°C for 10min; discard the supernatant, and store on ice 1000 μL of pre-cooled 70% ethanol, mix gently, wash once, centrifuge at 12,000 rpm at 4°C for 10 min; discard the supernatant, and air-dry; dissolve RNA with 20 μL of DEPC-treated triple distilled water, stor...
Embodiment 2
[0072] Example 2 Fluorescent quantitative PCR reaction condition optimization and standard curve making
[0073] The optimized fluorescent quantitative PCR reaction system is: 10 μL of 2×Premix Ex Taq (Probe qPCR), 0.8 μL of 10 μM upstream and downstream primers and probes, 1.0 μL of cDNA template, ddH 2 O supplemented to 20 μL.
[0074] The optimal fluorescent quantitative PCR reaction conditions are: 95°C for 30s; 95°C for 5s, 60°C for 30s, 40 cycles.
[0075] Determination of OD of standard recombinant positive plasmid 260nm and OD 280nm value and its ratio, calculate the plasmid concentration, and convert it into copy number, serially dilute it into 9 gradients with double distilled water 10 times (10 9 ~10 1 copies / μL). Using this as a template, a 20 μL reaction system was established, and amplified on a Bio-Rad fluorescent quantitative PCR instrument according to the optimized PCR reaction system. The obtained standard curve is shown in image 3 . As a result, the...
Embodiment 3
[0076] The sensitivity test of embodiment 3 real-time fluorescence quantitative PCR
[0077] The 10-fold serial dilution of SADS-CoV positive standard was used as a template for real-time fluorescent quantitative PCR (3.08×10 0 ~3.08×10 10 copies / μL, a total of 11 gradients, to determine their lower limit of detection. As a result, using the PMD19-T-N standard as a template, the detection limit of fluorescent quantitative PCR was 3.08×10 1 copies / μL (eg Figure 4 Shown), show that the fluorescent quantitative PCR primer and method of the present invention have high sensitivity.
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