Huang Qiuying, Chen Dongmei, Du Chen, Liu Qiaoqiao, Lin Su, Liang Lanlan, Xu Ye, Liao Yiqun, Li Qingge
Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen 361102, China.
Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen 361102, China;
Proc Natl Acad Sci U S A. 2022 Mar 1;119(9). doi: 10.1073/pnas.2110672119.
Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.
实时聚合酶链反应（Real-time PCR）是临床环境中使用最广泛的核酸检测工具。然而，目前的模式限制了每个反应中可检测的靶标数量。在此，我们描述了一种单步多重方法，能够在实时PCR热循环仪中每个反应检测数十个靶标。这种方法称为熔解阵列（MeltArray），它利用DNA聚合酶的5'-翼端核酸酶活性将一个介导探针切割成一个介导引物，该引物可以与一个分子信标报告分子结合，从而允许多个介导引物延伸，产生一系列具有每个靶标独特解链温度的荧光杂交体。使用多个用不同荧光团标记的分子信标报告分子，靶标的总数等于报告分子的数量乘以每个报告分子的介导引物数量。在各种情况下探索了熔解阵列的应用，包括在检测人类Y染色体微缺失的20重分析、确定血清型的62重分析、同时鉴定和定量呼吸道病原体的24重分析以及鉴定突变的微测序分析中，所有这些不同的分析都用临床样本进行了验证。由于其多重性、通用性、简单性和可及性的综合优点，熔解阵列方法应在临床环境中得到广泛应用。
