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首页> 美国卫生研究院文献>Medical Sciences >Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System
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Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System

机译：使用Luminex MultiCode ASRs系统对Roche Light Cycler 480 96孔板平台进行实时PCR测定和定量检测临床标本中的巨细胞病毒（CMV）的实施和验证

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Allogenic stem-cell therapies benefit patients in the treatment of multiple diseases; however, the side effects of stem-cell therapies (SCT) derived from the concomitant use of immune suppression agents often include triggering infection diseases. Thus, analysis is required to improve the detection of pathogen infections in SCT. We develop a polymerase chain reaction (PCR)-based methodology for the qualitative real-time DNA detection of cytomegalovirus (CMV), with reference to herpes simplex virus types 1 (HSVI), Epstein–Barr virus (EBV), and varicella-zoster virus (VZV) in blood, urine, solid tissues, and cerebrospinal fluid. This real-time PCR of 96-well plate format provides a rapid framework as required by the Food and Drug Administration (FDA) for clinical settings, including the processing of specimens, reagent handling, special safety precautions, quality control criteria and analytical accuracy, precisely reportable range (analyst measurement range), reference range, limit of detection (LOD), analytical specificity established by interference study, and analyte stability. Specifically, we determined the reportable range (analyst measurement range) with the following criteria: CMV copies ≥200 copies/mL; report copy/mL value; CMV copies ≤199 copies/mL; report detected but below quantitative range; CMV copies = 0 with report <200 copies/mL. That is, with reference range, copy numbers (CN) per milliliter (mL) of the LOD were determined by standard curves that correlated Ct value and calibrated standard DNA panels. The three repeats determined that the measuring range was 1E2~1E6 copies/mL. The standard curves show the slopes were within the range −2.99 to −3.65 with ≥ 0.98. High copy (HC) controls were within 0.17–0.18 log differences of DNA copy numbers; (2) low copy (LC) controls were within 0.17–0.18 log differences; (3) LOD was within 0.14–0.15 log differences. As such, we set up a fast, simple, inexpensive, sensitive, and reliable molecular approach for the qualitative detection of CMV pathogens. : This real-time PCR of the 96-well plate format provides a rapid framework as required by the FDA for clinical settings.
机译：同种异体干细胞疗法使患者受益于多种疾病的治疗。然而，伴随使用免疫抑制剂产生的干细胞疗法（SCT）的副作用通常包括引发感染性疾病。因此，需要进行分析以改善SCT中病原体感染的检测。我们针对巨细胞病毒（CMV）的定性实时DNA检测开发了基于聚合酶链反应（PCR）的方法，并参考了1型单纯疱疹病毒（HSVI），爱泼斯坦-巴尔病毒（EBV）和水痘带状疱疹血液，尿液，实体组织和脑脊液中的病毒（VZV）。这种96孔板格式的实时PCR提供了美国食品药品监督管理局（FDA）所要求的用于临床设置的快速框架，包括标本处理，试剂处理，特殊安全预防措施，质量控制标准和分析准确性，可精确报告的范围（分析物测量范围），参考范围，检测限（LOD），通过干扰研究确定的分析特异性以及分析物的稳定性。具体而言，我们根据以下标准确定了可报告范围（分析物测量范围）：CMV拷贝数≥200拷贝/ mL；报告拷贝/ mL值； CMV拷贝≤199拷贝/ mL;报告检测到但低于定量范围； CMV拷贝= 0，报告<200拷贝/ mL。也就是说，在参考范围内，LOD的每毫升（mL）拷贝数（CN）由与Ct值和校准的标准DNA板相关的标准曲线确定。 3次重复测定，测定范围为1E2〜1E6拷贝/ mL。标准曲线显示斜率在-2.99至-3.65范围内，≥0.98。高拷贝（HC）对照的DNA拷贝数差异在0.17-0.18对数内； （2）低拷贝（LC）对照的对数差异在0.17-0.18之间； （3）LOD在0.14-0.15 log对数内。因此，我们建立了一种快速，简单，廉价，敏感和可靠的分子方法，用于定性检测CMV病原体。 ：这种96孔板格式的实时PCR提供了FDA所要求的用于临床环境的快速框架。

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