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Taqman-MGB FQ-PCR for Human GPIIIa, GP1BA and PEAR1 SNPs by Hongxia Cheng, Yuhua Liu, Min Fu, Jingjing Liu, Gang Chen

Background: A reliable operation-convenient Taqman-MGB probe fluorescence quantity polymerase chain reaction (FQ-PCR) for detection human GPIIIa, GP1BA, and PEAR1 polymorphism were designed, and the performances were assessed.
Methods: Four sets of probes and primers for target alleles included rs5918 (176T>C), rs6065 (5792C>T), rs1204133 (2266G>A), and β-actin were designed, the reaction systems were optimized. Both artificial plasmids and clinical samples were tested by the research system and Sanger sequencing. The results were compared to assess the performance of the reaction system.
Results: The results shown the Taqman-MGB FQ-PCR could test as low as 5 ng/mL. There was no impact even if the concentration of DNA reached 170 ng/mL. The accuracy was 100% by detection of DNA samples and artificial plasmids. The coefficient of variation (CV) of 40 tests lasting 20 days was < 5% at low, medium, and high concentrations, respectively. Compared with Sanger sequencing, the AUC and Youden’s index of the reaction system were 0.991, 0.953 and 0.998, 0.993 for C/T allele of rs5918; 0.997, 0.976 and 0.997, 0.989 for T/C of rs6065; 0.998, 0.964, and 0.998, 0.976 for A/G of rs12041331, respectively. Eight kinds of biological ingredients in blood samples had no influence on the reaction system. The similar sequences and other mutant sites of three target gene sites would not impact or cross react with the reaction system. The performance of the system was stable for 11 months under -20℃ ± 5℃. The 275 clinical blood samples were tested by the research system and Sanger sequencing, the consistencies were 100%.
Conclusions: The research reaction system met the requirement in daily routine testing of laboratory medicine. This study was very meaningful for clinical rapid detection and personalized treatment.

DOI: 10.7754/Clin.Lab.2022.220132