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液基细胞学样本的自动化与手动DNA提取方法比较

Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples.

作者信息

Domínguez-Vigil Irma G, Barajas-Olmos Víctor H, Gallardo-Alvarado Lenny, Pérez-Maya Antonio A, Garza-Rodríguez Maria L, Magallanes-Garza Gerardo I, Cardona-Huerta Servando, Méndez-Lozano Daniel H, Vidal-Gutiérrez Oscar, Cantú De León David F, Barrera-Saldaña Hugo A

机构信息

Departamento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, NL, Mexico.

Laboratory for Translational Research, Rudy L. Ruggles Biomedical Research Institute, Western Connecticut Health Network, Danbury, Connecticut.

出版信息

Biopreserv Biobank. 2019 Dec;17(6):591-597. doi: 10.1089/bio.2018.0148. Epub 2019 Sep 26.

Abstract

Liquid-based cytology (LBC) has been used as a diagnostic tool for cervical cancer for years and is now being adopted for other gynecological cancers. LBC represents an important challenge to ensure that the process yields representative biospecimens for quality control (QC) of diagnostic procedures. In this study, we compare QC parameters (integrity, yield and purity, and polymerase chain reaction [PCR] amplification) of DNA isolated from LBC ( = 296) using two different nucleic acid isolation methods, manual ( = 233) or automated ( = 63). We also evaluated two different types of cytological brushes for sampling from the cervix. Our results suggest that manual isolation (yield 22.81 ± 1.92 μg) resulted in increased DNA recovery when compared with automated isolation (yield 9.96 ± 1.11 μg) from LBC samples, with a -value of <0.0003. We estimated that 98% (53/54) of the samples preserved the integrity of DNA and were suitable for standard molecular biology analyses. The β-globin gene was amplified in 100% (296/296) of the DNA samples by endpoint PCR. We found no significant difference between the performance of the cytological brushes ( value of <0.6711 in a general overview. However, when looking at the results from using each brush individually, the manual isolation method was statistically superior to the automated method. Our work illustrates the impact of good QC of preanalytic conditions, which will be important for the application of LBC for developing early detection methods for gynecological cancers.

摘要

液基细胞学检查(LBC)多年来一直被用作宫颈癌的诊断工具,目前正被应用于其他妇科癌症的诊断。LBC对于确保该过程能够产生用于诊断程序质量控制(QC)的代表性生物样本而言是一项重大挑战。在本研究中,我们比较了使用两种不同核酸分离方法(手动分离,n = 233;自动分离,n = 63)从LBC样本(n = 296)中分离的DNA的质量控制参数(完整性、产量和纯度以及聚合酶链反应[PCR]扩增)。我们还评估了两种不同类型的宫颈采样细胞学刷。我们的结果表明,与从LBC样本中自动分离DNA(产量为9.96±1.11μg)相比,手动分离(产量为22.81±1.92μg)可提高DNA回收率,P值<0.0003。我们估计98%(53/54)的样本保留了DNA的完整性,适合进行标准分子生物学分析。通过终点PCR,100%(296/296)的DNA样本中β-珠蛋白基因得到了扩增。我们发现细胞学刷的性能之间没有显著差异(总体P值<0.6711)。然而,单独查看每种刷子的使用结果时,手动分离方法在统计学上优于自动分离方法。我们的工作说明了分析前条件良好质量控制的影响,这对于LBC在妇科癌症早期检测方法开发中的应用至关重要。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f75f/6921247/042702ef3230/bio.2018.0148_figure1.jpg

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