Hebei Medical University, Shijiazhuang, Hebei, China.
NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping District, Beijing, China.
Braz J Med Biol Res. 2020 Nov 27;54(2):e9549. doi: 10.1590/1414-431X20209549. eCollection 2020.
Single nucleotide polymorphisms (SNPs) have important application value in the research of population genetics, hereditary diseases, tumors, and drug development. Conventional methods for detecting SNPs are typically based on PCR or DNA sequencing, which is time-consuming, costly, and requires complex instrumentation. In this study, we present a duplex probe-directed recombinase amplification (duplex-PDRA) assay that can perform real-time detection of two SNPs (rs6983267 and rs1447295) in four reactions in two tubes at 39°C within 30 min. The sensitivity of duplex-PDRA was 2×103-104 copies per reaction and no cross-reactivity was observed. A total of 382 clinical samples (179 prostate cancer patients and 203 controls) from northern China were collected and tested by duplex-PDRA assay and direct sequencing. The genotyping results were completely identical. In addition, the association analysis of two SNPs with prostate cancer risk and bone metastasis was conducted. We found that the TT genotype of rs6983267 (OR: 0.42; 95%CI: 0.23-0.78; P=0.005) decreased the risk of prostate cancer, while the CA genotype of rs1447295 (OR: 1.89; 95%CI: 1.20-2.96; P=0.005) increased the risk of prostate cancer. However, no association between the two SNPs (rs6983267 and rs1447295) and bone metastasis in prostate cancer was found in this study (P>0.05). In conclusion, the duplex-PDRA assay is an effective method for the simultaneous detection of two SNPs and shows great potential for widespread use in research and clinical settings.
单核苷酸多态性 (SNP) 在群体遗传学、遗传性疾病、肿瘤和药物开发的研究中具有重要的应用价值。传统的 SNP 检测方法通常基于 PCR 或 DNA 测序，这些方法既耗时又昂贵，并且需要复杂的仪器。在本研究中，我们提出了一种双探针引导重组酶扩增（duplex-PDRA）测定法，该方法可在 39°C 下在两个管中进行四个反应，在 30 分钟内实时检测两个 SNP（rs6983267 和 rs1447295）。duplex-PDRA 的灵敏度为每个反应 2×103-104 拷贝，没有观察到交叉反应。共收集并测试了来自中国北方的 382 例临床样本（179 例前列腺癌患者和 203 例对照），采用 duplex-PDRA 测定法和直接测序。基因分型结果完全一致。此外，还进行了两项 SNP 与前列腺癌风险和骨转移关联分析。我们发现 rs6983267 的 TT 基因型（OR：0.42；95%CI：0.23-0.78；P=0.005）降低了前列腺癌的风险，而 rs1447295 的 CA 基因型（OR：1.89；95%CI：1.20-2.96；P=0.005）增加了前列腺癌的风险。然而，在本研究中未发现这两个 SNP（rs6983267 和 rs1447295）与前列腺癌骨转移之间存在关联（P>0.05）。总之，duplex-PDRA 测定法是一种同时检测两个 SNP 的有效方法，在研究和临床环境中具有广泛应用的潜力。
