Köller Thomas, Kurze Daniel, Lange Mirjam, Scherdin Martin, Podbielski Andreas, Warnke Philipp
Institute of Medical Microbiology, Virology, and Hygiene, Rostock University Hospital, Rostock, Germany.
PLoS One. 2016 Apr 19;11(4):e0153991. doi: 10.1371/journal.pone.0153991. eCollection 2016.
A fully automated multiplex real-time PCR assay--including a sample process control and a plasmid based positive control--for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88-258) copies/ml for HSV1, 171 (CI 95%, 148-194) copies/ml for HSV2 and 84 (CI 95%, 5-163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.
在BD Max平台上开发了一种全自动多重实时PCR检测方法，包括样本过程控制和基于质粒的阳性对照，用于从脑脊液（CSF）中检测和区分单纯疱疹病毒1型（HSV1）、单纯疱疹病毒2型（HSV2）和水痘-带状疱疹病毒（VZV）。将该方法的性能与使用easyMAG和LightCycler 480/II的既定认可多重实时PCR方案进行了比较，这两种都是病毒分子诊断中非常常见的设备。为了进行临床验证，用这两种方法检测了123份脑脊液标本和40份来自全国实验室间比对的参考样本，脑脊液样本和参考样本的一致性分别为97.6%和100%。使用BD Max平台显示，HSV1的灵敏度为173（95%置信区间，88 - 258）拷贝/毫升，HSV2为171（95%置信区间，148 - 194）拷贝/毫升，VZV为84（95%置信区间，5 - 163）拷贝/毫升。通过检测25种常见的人类病毒、细菌和真菌病原体可排除交叉反应。工作流程分析显示，该检测方法的测试时间更短，准备步骤显著减少且更简便，有可能降低手动评估患者样本时出现的错误率。该方案允许在BD Max平台上进行全自动PCR检测，以同时检测脑脊液标本中的疱疹病毒科。由HSV1、HSV2和VZV引起的单一或多重感染能够以良好的灵敏度可靠区分。检测方法中包含控制参数，因此使其适用于当前质量管理要求。
