A real-time fluorescent quantitative PCR primer, probe and kit for detecting tree shrew adenovirus
A technology of real-time fluorescence quantification and kits, applied in biochemical equipment and methods, microorganisms, and methods based on microorganisms, etc., can solve the problems of inability to quantify the copy number of DNA or RNA, and achieve cost saving, high sensitivity, and high accuracy sexual effect
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Embodiment 1
[0046] Example 1 Design of special primers and probes for tree shrew adenovirus
[0047] Designed and synthesized according to the tree shrew whole genome sequence (GenBank: MF780605) which was isolated, identified and sequenced by our laboratory. The amplicon size is 122bp. The 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected Has a fluorescent quencher. Using the primer for PCR amplification can realize qualitative and quantitative detection with good specificity, high sensitivity and strong accuracy. Wherein the preferred primer and probe sequences are:
[0048] Upstream primer: 5′-AAAGAATGCCCACCTCCCAT-3′; (SEQ ID NO.1)
[0049] Downstream primer: 5′-CAGTGGCAGGCCATTAAGC-3′; (SEQ ID NO.2)
[0050] Probe primer: 5′-Fam-CTCGCCGCCGCCGGTTTCAC-Tamra-3′. (SEQ ID NO.3)
[0051] After homology comparison with the designed primers and probes, it was found that it was only highly conserved against tree shrew adenovirus, and there was ...
Embodiment 2
[0052] Example 2 Preparation of standard positive template
[0053] The positive tree shrew adenovirus DNA preserved in our laboratory was used as a template to amplify the target gene fragment.
[0054] Reaction system: Gold Mix (green) 20 μL, 10 μM upstream primer 1 μL, 10 μM downstream primer 1 μL, 50ng / μL DNA template 3 μL, final volume 25 μL.
[0055] The reference annealing temperature of the kit is 55-60°C, and the optimized reaction conditions are: 98°C for 2 min; 98°C for 10 s, 58°C for 15 s, 72°C for 15 s, a total of 35 cycles; 72°C for 5 min.
[0056] The amplified product was identified by agarose gel electrophoresis with a volume fraction of 1.5%; the gel was recovered and purified; the target gene fragment was ligated with the PUC57 vector; the ligated product was transformed into DH5α competent cells; the sequencing verification was the standard positive template. The absorbance values of A260 and A280 were measured by ultraviolet spectrophotometer, the ratio...
Embodiment 3
[0058] Example 3 Establishment of Real-time Fluorescent Quantitative PCR Amplification Method
[0059] 1. Real-time fluorescence quantitative PCR template preparation - sample total DNA extraction:
[0060] 1) Add 500 μL of lysate (100 mmol / L Tris-Cl pH=8.5, 0.1mol / L EDTA, 0.5% SDS) to 200 μL of the solution to be tested (if the sample is a tissue that needs to be ground and homogenized), mix well by inverting , 5min.
[0061] 2) Add 6 μL of proteinase K, bathe in water at 55°C for 15 minutes, and cool down.
[0062] 3) Add 700 μL of a mixed solution of phenol, chloroform and isoamyl alcohol (25:24:1 by volume), and shake to mix.
[0063] 4) Centrifuge at 10000rpm / min for 5min to take the supernatant.
[0064] 5) Repeat steps 3 and 4, then pool the supernatants.
[0065] 6) Add 2 times the volume of the combined supernatant, pre-cooled to 4°C absolute ethanol and 1 / 10 volume of the combined supernatant 3mol / L sodium acetate solution.
[0066] 7) Precipitate at -20°C for 1...
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