Double-color competitiveness fluorescent quantitation polymerase chain reaction detection kit
A fluorescence quantitative and two-color fluorescence technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of complex technology, inability to realize automation and high-throughput detection, and long time consumption
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Embodiment 1
[0013] 1. Design of primers and probes
[0014] According to the sequence of DSCR1[gi:7768679] in genebank, the primers of DSCR were designed as follows:
[0015] Upstream primer: 5'-AAA GTT TCT TCT GGA TCT ACA G-3' (SEQ ID NO: 1);
[0016] Downstream primer: 5'-TCC TCT GTG CTC TGA GCT AAG-3' (SEQ ID NO: 2);
[0017] DSCR-specific TaqMan probe DSCRTM: 5'-[FAM]ACA TTT TGG ATG CAC TGG GA[TAMRA]-3' (SEQ ID NO: 3); USC2-specific TaqMan probe 5'-[HEX]CAA AAC TCA CAGAGG GAC TC[TAMRA]-3' (SEQ ID NO: 4).
[0018] DSCR amplification product 171bp, the sequence is: AAAAAATTTTTTTTTTT CAATCTAAAA ACTGGAAATT CTAGGGTTTT TGTACATTTTGGATGCACTG GGAATTTATT AGCACAAAAT CATTCTTTGC AACTCAAAATTCAGAAGGGA CTCTACCATAT (SEQ ID NO: 5).
[0019] The USC2 amplification product is 169bp, and the sequence is: GAGTTTTTCATTTCCAAT CTAAAAACTA GAAGCTCTAG CATTTTGTACA TTTTTTGTTGTTGCACTGGAA GTTTAACTATT GGCACAAAAT CATTCTTCAAA ACTCACAGAGGGACTCTGCC ATTA (SEQ ID NO: 6).
[0020] 2. Two-color competitive fluore...
Embodiment 2
[0027] 1. Design of primers and probes
[0028] Upstream primer: 5'-AAA GTT TCT TCT GGA TCT ACA G-3' (SEQ ID NO: 1); Downstream primer: 5'-TCC TCT GTG CTC TGA GCT AAG-3' (SEQ ID NO: 2); DSCR TaqMan probe for USC2: 5'-[FAM] ACA TTT TGG ATG CAC TGG GA[TAMRA]-3' (SEQ ID NO: 3); TaqMan probe for USC2: 5'-[HEX]CAA AAC TCA CAG AGG GAC TC[TAMRA]-3' (SEQ ID NO: 4).
[0029] 2. Two-color competitive fluorescent quantitative PCR reaction
[0030] Mix 50ul reaction system, containing 15pmol each of the two primers, 15pmol of each probe, 500ng of patient or normal human DNA, 1X PCR buffer, 2u of Taq DNA polymerase, final concentration of Mg++ 2mmol / L, final concentration of dNTPs 0.4mmol / L and then Pre-denature at 96°C for 2 min on a FTC2000 real-time fluorescent quantitative PCR instrument, then denature at 94°C for 10 sec, anneal at 52°C for 30 sec, and extend at 60°C for 40 sec, for a total of 45 cycles, and take pictures at 60°C. Experiments were repeated three times for each sampl...
Embodiment 3
[0036] 1. Design of primers and probes
[0037] Upstream primer: 5'-AAA GTT TCT TCT GGA TCT ACA G-3' (SEQ ID NO: 1); Downstream primer: 5'-TCC TCT GTG CTC TGA GCT AAG-3' (SEQ ID NO: 2); DSCR TaqMan probe for USC2: 5'-[HEX]CAA AAC TCA CAG AGG GAC TC[TAMRA]-3' (SEQ ID NO: 4).
[0038] 2. Two-color competitive fluorescent quantitative PCR reaction
[0039] Mix 50ul reaction system, containing 20pmol each of the two primers, 20pmol of each probe, 1ug of patient or normal human DNA, 1.5X PCR buffer, 3u of Taq DNA polymerase, final concentration of Mg++ 3.5mmol / L, final concentration of dNTPs 0.5mmol / L L was then pre-denatured on a FTC2000 real-time fluorescent quantitative PCR instrument at 96°C for 2 min, then denatured at 94°C for 10 sec, annealed at 55°C for 30 sec, and extended at 60°C for 40 sec, a total of 45 cycles, and photographed at 60°C. Experiments were repeated three times for each sample.
[0040] 3. Standard curve of fluorescent competitive quantitative PCR
...
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