Primers, probes and kit for detecting 19-Del mutation of epidermal growth factor receptor (EGFR) gene
A kit and gene technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of insufficient sensitivity, low sensitivity, and inability to meet clinical needs, so as to ensure sensitivity and specificity, Consistent detection sensitivity and improved amplification efficiency
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Embodiment 1
[0046] Example 1: The primers and probes for detecting 19-Del were synthesized by Bailige Biotechnology Co., Ltd., as shown in the following table:
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Embodiment 2
[0050] Example 2: Synthesis of positive mutant plasmids used to test each mutant primer. The mutant type that can be amplified with the same specific upstream primer only needs to synthesize one of the plasmids, and the insert sequence is designed and provided by Shanghai Sangon Bioengineering Co., Ltd. Synthesis, a total of 35 positive plasmids (pUC57 cloning plasmid + insert sequence), the specific primer positions match 53 common mutation types, covering more than 99% of the mutation incidence rate, as shown in the following table:
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[0056] Note: *The mutation corresponding ID number and mutation incidence data come from the world's most comprehensive public tumor mutation database (https: / / cancer.sanger.ac.uk / cosmic)
Embodiment 3
[0057] Example 3: Preparation of EGFR gene 19-Del mutation detection kit.
[0058] (1) 2*supermix: The PCR reaction solution contains hot start DNA polymerase, Mg 2+ , PCR reaction buffer, dNTPmixture, betaine and tris(2-carboxyethyl)phosphine (TCEP), BSA, etc. Preferably, the concentration of hot-start DNA polymerase is 0.02-0.04U / μL, the concentration of Mg2+ is 7-8mM, the concentration of dNTP mixture is 500-1000μM, the concentration of betaine is 0.5-1M, and the concentration of TCEP is 0.5-1mM. Store at -20°C.
[0059] (2) Mutant primer-probe master mix (20*MUT): Dissolve the primers and probes shown in SEQ ID NO: 1-34 in double distilled water, the concentration of each primer is 100 μM, the concentration of the probe 10 μM, prepare 20*MUT premix; preferably, the concentration of primers 22 and 25 is 3-4 μM, the concentration of primers 1 and 32 is 7-8 μM, and the concentration of the remaining 29 primers is 1-2 μM, Probe No. 34 has a concentration of 4-6 μM and is st...
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