一种新的全自动从全血样本中提取定量实时 PCR 的系统：免疫抑制患者机会性感染的常规监测。

A new highly automated extraction system for quantitative real-time PCRs from whole blood samples: routine monitoring of opportunistic infections in immunosuppressed patients.

机构信息

Department of Virology, CH Toulouse, France.

出版信息

J Clin Virol. 2012 Apr;53(4):314-9. doi: 10.1016/j.jcv.2012.01.002. Epub 2012 Jan 30.

DOI:10.1016/j.jcv.2012.01.002

PMID:22296792

Abstract

BACKGROUND

Rapid, high throughput extraction systems are needed to monitor viral infections in immunosuppressed patients.

OBJECTIVES

Evaluate the performance of the MagNA Pure 96™ extraction system, and compare it to the COBAS Ampliprep™ for quantitative real-time PCR from whole blood samples.

STUDY DESIGN

Compare the MagNA Pure LC™, COBAS Ampliprep™ and MagNA Pure 96™ using ten-fold dilutions of blood samples containing cytomegalovirus. Evaluate analytical performances of the MagNA Pure 96™ from test samples containing cytomegalovirus. Evaluate clinical performances from 209 blood samples collected prospectively, extracted with the COBAS Ampliprep™ and the MagNA Pure 96™ systems and tested for cytomegalovirus, Epstein-Barr, BK and JC viruses.

RESULTS

All three extraction systems gave similar results with dilutions of a cytomegalovirus-positive sample. Analytical tests showed that the limit of detection was 500 copies/ml, specificity was 100%, with no cross-contamination. Quantification was linear from 3.0 to 6.0 log(10)copies/ml. Intra-assay variation was 8.3-0.9% and inter-assay variation 8.8-5.2%. Clinical specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments agreed well for cytomegalovirus (r=0.54; p=0.07), Epstein-Barr virus (0.69; p=0.0005) and BK virus (0.85; p=0.01). All 55 samples were negative for JC virus. Mean loads were similar for cytomegalovirus (0.17 log(10)copies/ml) and BK virus (-0.24 log(10)copies/ml) while that of Epstein-Barr virus was slightly lower (1.02 log(10)copies/ml).

CONCLUSIONS

The MagNA Pure 96™ instrument is an easy-to-use, reliable high throughput platform for extracting nucleic acid from clinical whole blood specimens.

摘要

背景

需要快速、高通量的提取系统来监测免疫抑制患者的病毒感染。

目的

评估 MagNA Pure 96 提取系统的性能，并将其与 COBAS Ampliprep 实时定量 PCR 从全血样本进行比较。

研究设计

使用包含巨细胞病毒的血液样本十倍稀释液比较 MagNA Pure LC、COBAS Ampliprep 和 MagNA Pure 96。评估 MagNA Pure 96 从包含巨细胞病毒的测试样本中的分析性能。从 209 份前瞻性采集的血液样本中评估临床性能，这些样本用 COBAS Ampliprep 和 MagNA Pure 96 系统提取并检测巨细胞病毒、EB 病毒、BK 病毒和 JC 病毒。

结果

三种提取系统对巨细胞病毒阳性样本的稀释液均给出相似的结果。分析测试表明，检测限为 500 拷贝/ml，特异性为 100%，无交叉污染。定量从 3.0 到 6.0 log(10)拷贝/ml 呈线性。批内变异为 8.3-0.9%，批间变异为 8.8-5.2%。用 MagNA Pure 96 和 COBAS Ampliprep 仪器提取的临床标本对巨细胞病毒（r=0.54；p=0.07）、EB 病毒（0.69；p=0.0005）和 BK 病毒（0.85；p=0.01）的检测结果一致。所有 55 份样本均为 JC 病毒阴性。巨细胞病毒（0.17 log(10)拷贝/ml）和 BK 病毒（-0.24 log(10)拷贝/ml）的平均负荷相似，而 EB 病毒的负荷略低（1.02 log(10)拷贝/ml）。