The primers were designed based on published gene sequences of Homo sapiens (NCBI Reference Sequence Database) using NCBI’s Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 3 September 2025). All primers were synthesized by Tsingke (Changsha, China) and validated via PCR a two-step quality control process. PCR amplification followed by Sanger sequencing to confirm target specificity, and gradient PCR optimization to determine optimal annealing temperatures.
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