用于检测肠道病毒RNA的自动化和手动核酸提取方法的比较。
Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA.
作者信息
Knepp Julia H, Geahr Melissa A, Forman Michael S, Valsamakis Alexandra
机构信息
Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-7093, USA.
出版信息
J Clin Microbiol. 2003 Aug;41(8):3532-6. doi: 10.1128/JCM.41.8.3532-3536.2003.
Automated nucleic acid extraction is an attractive alternative to labor-intensive manual methods. We compared two automated methods, the BioRobot M48 instrument (Qiagen, Inc.) and MagNA Pure (Roche Applied Sciences) methods, to two manual methods, the QIAamp Viral RNA Mini kit (Qiagen) and TRIzol (Invitrogen), for the extraction of enterovirus RNA. Analytical sensitivity was assessed by dilution analysis of poliovirus type 2 Sabin in cerebrospinal fluid. The sensitivity of PCR was equivalent after RNA extraction with QIAamp, BioRobot M48, and MagNA Pure. All 18 replicates of 100 PFU/ml were detected after extraction by the four methods. Fewer replicates of each successive dilution were detected after extraction by each method. At 10(-1) PFU/ml, 17 of 18 replicates were positive by QIAamp, 15 of 18 replicates were positive by BioRobot M48, and 12 of 18 replicates were positive by MagNA Pure; at 10(-2) PFU/ml, 4 of 17 replicates were positive by QIAamp, 2 of 18 replicates were positive by BioRobot M48, and 0 of 18 replicates were positive by MagNA Pure. At 10(-3) PFU/ml, no replicates were detected. Evaluation of TRIzol was discontinued after nine replicates due to a trend of lower sensitivity (at 10(-3) PFU/ml eight of nine replicates were positive at 100 PFU/ml, four of nine replicates were positive at 10(-1) PFU/ml, and zero of nine replicates were positive at 10(-2) PFU/ml). Concordant results were obtained in 24 of 28 clinical specimens after extraction by all methods. No evidence of contamination was observed after extraction by automated instruments. The data indicate that the sensitivity of enterovirus PCR is largely similar after extraction by QIAamp, BioRobot M48, and MagNA Pure; a trend of decreased sensitivity was observed after TRIzol extraction. However, the results of enterovirus PCR were largely concordant in patient samples, indicating that the four extraction methods are suitable for detection of enteroviruses in clinical specimens.
自动化核酸提取是一种有吸引力的替代劳动密集型手工方法的技术。我们将两种自动化方法，即BioRobot M48仪器（Qiagen公司）和MagNA Pure（罗氏应用科学公司）方法，与两种手工方法，即QIAamp病毒RNA迷你试剂盒（Qiagen）和TRIzol（Invitrogen），用于肠道病毒RNA的提取进行了比较。通过对脑脊液中2型脊髓灰质炎病毒Sabin株进行稀释分析来评估分析灵敏度。用QIAamp、BioRobot M48和MagNA Pure提取RNA后，PCR的灵敏度相当。四种方法提取后，所有18份100 PFU/ml的重复样本均被检测到。每种方法提取后，每个连续稀释度的重复样本检测到的数量减少。在10⁻¹ PFU/ml时，QIAamp提取的18份重复样本中有17份呈阳性，BioRobot M48提取的18份重复样本中有15份呈阳性，MagNA Pure提取的18份重复样本中有12份呈阳性；在10⁻² PFU/ml时，QIAamp提取的17份重复样本中有4份呈阳性，BioRobot M48提取的18份重复样本中有2份呈阳性，MagNA Pure提取的18份重复样本中有0份呈阳性。在10⁻³ PFU/ml时，未检测到重复样本。由于灵敏度较低的趋势（在100 PFU/ml时，9份重复样本中有8份呈阳性；在10⁻¹ PFU/ml时，9份重复样本中有4份呈阳性；在10⁻² PFU/ml时，9份重复样本中有0份呈阳性），在9份重复样本后停止了对TRIzol的评估。所有方法提取后，28份临床样本中有24份获得了一致的结果。自动化仪器提取后未观察到污染迹象。数据表明，用QIAamp、BioRobot M48和MagNA Pure提取后，肠道病毒PCR的灵敏度大致相似；TRIzol提取后观察到灵敏度下降的趋势。然而，患者样本中肠道病毒PCR的结果大致一致，表明这四种提取方法适用于临床样本中肠道病毒的检测。
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