Primer group, reagent and/or kit and system for detecting lung cancer chemotherapy related genes, and application
A technology of primer sets and kits, which is applied in the field of primer sets for detection of lung cancer chemotherapy-related genes, can solve the problems of inability to detect unknown mutations, low throughput of in situ hybridization, and expensive probes, achieving short detection cycles, The effect of easy analysis and high sensitivity
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Embodiment 1
[0047] Example 1 Primer Design Optimization
[0048] 1) Primer sequence optimization
[0049] According to the site in Table 1, the present invention comprehensively designs PCR primers and single-base extension primers based on software and design experience, uses gDNA as a template to detect the working efficiency of primers, and finally screens out the best PCR primers and single-base extension primers through multiple rounds of design and repeated optimization. For base extension primers, see the primer sequences in Tables 2 and 3 for details.
[0050] Table 2. PCR primer sequences
[0051]
[0052]
[0053] Table 3. Single-point extension primer sequences
[0054]
[0055]
[0056] 2) Optimization of annealing temperature system
[0057] The annealing temperature is an important parameter in the reaction system. During the experiment, it was found that when the annealing temperature of the PCR reaction was set at 56°C as usual, the amplification efficiency ...
Embodiment 2
[0058] Embodiment 2 system verification
[0059] The verification of the system of the present invention includes accuracy, specificity, sensitivity, precision and comparison between personnel, etc., specifically:
[0060] The accuracy verification scheme of the present invention: each site detects 20 cases, compared with Sanger sequencing, the expected target is 95%.
[0061] The specificity verification scheme of the present invention: included in the accuracy, the expected target is 95%.
[0062] The sensitive verification scheme of the present invention: included in the accuracy, the expected target is 95%.
[0063] The precision verification scheme of the present invention (including intra-batch, inter-batch, and personnel comparison, not involving inter-instrument comparison) has an expected target of 95%.
[0064] Intra-assay precision: each sample was repeated 3 times in the same batch, and the intra-assay precision was compared:
[0065] Batch-to-batch precision: t...
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