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Comparison of Automated Nucleic Acid Extraction Methods with Manual Extraction

机译:自动核酸提取方法与手动提取方法的比较

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摘要

Automated nucleic acid extractors can improve workflow and decrease variability in the clinical laboratory. We evaluated Qiagen EZ1 (Valencia, CA) and bioMérieux (Durham, NC) easyMAG extractors compared with Qiagen manual extraction using targets and matrices commonly available in the clinical laboratory. Pooled samples were spiked with various organisms, serially diluted, and extracted in duplicate. The organisms/matrices were Bordetella pertussis/bronchoalveolar lavage, herpes simplex virus II/cerebrospinal fluid, coxsackievirus A9/cerebrospinal fluid, BK virus/plasma, and Mycoplasma pneumoniae/endotracheal tube samples. Extracts were amplified in duplicate using real-time PCR assays, and amplification of the target at a cycle threshold of 35 using the manual method was used for comparison. Amplification efficiency of nucleic acids extracted by automated methods was similar to that by the manual method except for a loss of efficiency for M. pneumoniae in endotracheal tube samples. The EZ1 viral kit 2.0 gave better results for coxsackievirus A9 than the EZ1 viral kit version 1.0. At the lowest limit of detection (past a cycle threshold of 35), the easyMAG was more likely to produce amplifiable nucleic acid than were either the EZ1 or manual extraction. Operational complexity, defined as the number of manipulations required to obtain an extracted sample, was the lowest for the easyMAG. The easyMAG was the most expensive of the methods, followed by the EZ1 kit and manual extraction.
机译:自动化核酸提取器可改善临床实验室的工作流程并减少变异性。我们将Qiagen EZ1(巴伦西亚,加利福尼亚州)和bioMérieux(杜伦,北卡罗来纳州)easyMAG提取器与使用临床实验室中常见的目标物和基质进行手动Qiagen提取相比进行了评估。将合并的样品掺入各种生物,进行连续稀释,并重复提取两次。生物体/基质是百日咳博德特氏菌/支气管肺泡灌洗,单纯疱疹病毒II /脑脊液,柯萨奇病毒A9 /脑脊液,BK病毒/血浆和肺炎支原体/气管导管样品。使用实时PCR分析将提取物一式两份扩增,并使用手动方法以35循环阈值扩增靶标进行比较。通过自动化方法提取的核酸的扩增效率与通过人工方法获得的核酸的扩增效率相似,只是气管内插管样品中的肺炎支原体效率降低。 EZ1病毒试剂盒2.0对柯萨奇病毒A9的效果比EZ1病毒试剂盒1.0版更好。在最低检测限(过去的循环阈值为35)下,与EZ1或手动提取相比,easyMAG更有可能产生可扩增的核酸。操作复杂度(定义为获取提取样本所需的操作次数)是easyMAG最低的。 easyMAG是最昂贵的方法,其次是EZ1试剂盒和手动提取。

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