基于多重不对称实时荧光定量PCR-HRM生物传感同时检测基因亚甲基四氢叶酸还原酶和甲硫氨酸合酶还原酶的三种基因型,Analytical Chemistry

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基于多重不对称实时荧光定量PCR-HRM生物传感同时检测基因亚甲基四氢叶酸还原酶和甲硫氨酸合酶还原酶的三种基因型
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-12 , DOI: 10.1021/acs.analchem.2c02096
Wen Yu ₁ , Juan Yao ₂ , Zhang Zhang _{3,

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Affiliation

叶酸代谢基因的基因分型在疾病诊断和预防中具有重要意义。然而，目前用于叶酸代谢基因分型的检测方法大多基于测序和芯片，成本高，过程费时费力。在此，我们报道了一种多重不对称 PCR-HRM 策略，用于在单管中鉴定叶酸代谢基因的基因型。拟议的多重 PCR-HRM 测定已成功应用于鉴定叶酸代谢基因、亚甲基四氢叶酸还原酶（C677T、A1298C）和甲硫氨酸合酶还原酶 A66G 的基因型，在 1 μL 基因组 DNA (gDNA) 样本上直接从血液样本中释放，基因分型结果与测序结果100%一致。该测定使我们能够准确检测 gDNA 的基因型，检测限低至 1 ng，符合临床要求。更重要的是，多重不对称 PCR-HRM 生物传感的抗气溶胶污染能力首先得到解决，并得到评估，因为它可以承受大约 12.5-25% 的干扰核酸污染。由于具有多重检测、高精度、抗气溶胶污染和无需开管程序等优点，该方法将为叶酸代谢基因分型和临床诊断中的其他 SNP 基因分型建立快速且具有成本效益的平台铺平道路. 多重不对称 PCR-HRM 生物传感的抗气溶胶污染能力首先得到解决，并得到评估，因为它可以承受大约 12.5-25% 的干扰核酸污染。由于具有多重检测、高精度、抗气溶胶污染和无需开管程序等优点，该方法将为叶酸代谢基因分型和临床诊断中的其他 SNP 基因分型建立快速且具有成本效益的平台铺平道路. 多重不对称 PCR-HRM 生物传感的抗气溶胶污染能力首先得到解决，并得到评估，因为它可以承受大约 12.5-25% 的干扰核酸污染。由于具有多重检测、高精度、抗气溶胶污染和无需开管程序等优点，该方法将为叶酸代谢基因分型和临床诊断中的其他 SNP 基因分型建立快速且具有成本效益的平台铺平道路.

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Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Genotyping of folate metabolism genes is of great importance in disease diagnosis and prevention. However, most current detection methods used for folate metabolism gene genotyping are based on sequencing and chips, which suffer from a high cost and laborious and time-consuming procedures. Herein, we reported a multiplex asymmetric PCR-HRM strategy for identifying genotypes of folate metabolism genes in a single tube. The proposed multiplex PCR-HRM assay has been successfully applied to identify the genotypes of folate metabolism genes, methylene tetrahydrofolate reductase (C677T, A1298C) and methionine synthase reductase A66G, on 1 μL of genomic DNA (gDNA) samples directly released from blood specimens, and the genotyping results were 100% consistent with the results of sequencing. The assay allows us to accurately detect the genotypes of gDNA with the detection limit down to 1 ng, which meets the clinical requirement. What is more, the capacity of resistance to aerosol pollution of the multiplex asymmetric PCR-HRM biosensing was first addressed and has been evaluated as it can withstand contamination of roughly 12.5–25% interfering nucleic acids. Because of the advantages of multiplex detection, high accuracy, and resistance to aerosol pollution and having no open tube procedure, this approach would pave the way for establishing a fast and cost-effective platform for folate metabolism gene genotyping and other SNP genotyping in clinical diagnostics.

更新日期：2022-09-12

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