Taqman-MGB FQ-PCR 用于人 GPIIIa、GP1BA 和 PEAR1 SNPs 的检测。

Clin Lab. 2022 Oct 1;68(10). doi: 10.7754/Clin.Lab.2022.220132.

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.

背景

设计了一种可靠、操作简便的 Taqman-MGB 探针荧光定量聚合酶链反应（FQ-PCR），用于检测人 GPIIIa、GP1BA 和 PEAR1 多态性，并评估了其性能。

方法

设计了针对目标等位基因 rs5918（176T>C）、rs6065（5792C>T）、rs1204133（2266G>A）和β-肌动蛋白的四组探针和引物，优化了反应体系。通过研究系统和 Sanger 测序对人工质粒和临床样本进行了检测，并对结果进行了比较，以评估反应体系的性能。

结果

结果表明，Taqman-MGB FQ-PCR 可检测低至 5ng/mL 的 DNA。即使 DNA 浓度达到 170ng/mL 也没有影响。通过对 DNA 样本和人工质粒的检测，该方法的准确率达到 100%。在低、中、高浓度下，40 次连续 20 天的检测，CV 值分别<5%。与 Sanger 测序相比，该反应系统的 AUC 和 Youden 指数分别为 rs5918 位点 C/T 等位基因的 0.991、0.953 和 0.998、0.989；rs6065 位点 T/C 的 0.997、0.976 和 0.997、0.989；rs12041331 位点 A/G 的 0.998、0.964 和 0.998、0.976。血液样本中的 8 种生物成分对反应体系无影响。三个目标基因位点的相似序列和其他突变位点不会对反应体系产生影响或交叉反应。在-20℃±5℃条件下，该系统的性能稳定 11 个月。通过研究系统和 Sanger 测序检测了 275 例临床血液样本，两者的一致性为 100%。