Diagnosis and quantitative evaluation of parvovirus B19 infections by real‐time PCR in the clinical laboratory
Diagnosis and quantitative evaluation of parvovirus B19 infections by real‐time PCR in the clinical laboratory
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临床实验室实时荧光定量PCR对细小病毒B19感染的诊断和定量评估
DOI：
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发表时间：
2002
影响因子：
12.7
通讯作者：
M. Musiani
中科院分区：
文献类型：
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作者：
E. Manaresi;G. Gallinella;E. Zuffi;F. Bonvicini;M. Zerbini;M. Musiani
开发了一种真实的实时PCR检测方法，用于定量检测临床血清样本中的B19 DNA。使用LightCycler仪器进行测定，并使用荧光双链DNA结合染料SYBR绿色I连续监测产物形成。通过优化的PCR方案，该系统能够将靶DNA定量至3 × 101个基因组拷贝/反应，并检测低至3 × 100个基因组拷贝/反应。使用真实的PCR检测108份临床疑似B19感染患者的血清样本中的B19 DNA，与标准化PCR-ELISA相比，灵敏度为92.7%，特异性为100%。使用LightCycler检测试剂盒，检测和定量临床血清样本中的B19 DNA的整个过程耗时长达90分钟，比PCR-ELISA快5倍。通过真实的时间PCR对阳性样本中的B19 DNA进行定量，结果显示B19感染急性活动期样本中的平均B19 DNA拷贝数为1.1 × 109/ml（DNA+、IgM+、IgG−），活动期样本中4.3 × 106 B19 DNA拷贝/ml（DNA+、IgM+、IgG+），长期活动期样本中3.7 × 105基因组拷贝/ml（DNA+，IgM-，IgG+）在急性活动期血清组和急性活动期血清组之间，B19 DNA含量在统计学上显著降低，B19感染。LightCycler技术为B19 DNA的检测和定量提供了高水平的灵敏度、特异性和快速性，代表了B19感染实验室诊断的显著改进。J. Med. Virol. 67：275-281，2002.© 2002 Wiley利斯公司
A real‐time PCR assay was developed for quantitative detection of B19 DNA in clinical serum samples. The assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double‐stranded DNA binding dye SYBR Green I. With an optimized PCR protocol, this system was able to quantitate the target DNA down to 3 × 101 genome copies/reaction and to detect as few as 3 × 100 genome copies/reaction. Real‐time PCR was used to detect B19 DNA in 108 serum samples from patients with a clinical suspicion of B19 infection, showing a sensitivity of 92.7% and a specificity of 100% when compared with a standardized PCR‐ELISA considered as the standard. Using the LightCycler assay, the entire procedure of detection and quantitation of B19 DNA in clinical serum samples took up to 90 min proving five times faster than PCR‐ELISA. B19 DNA quantitation in positive samples by real‐time PCR showed a mean of 1.1 × 109 B19 DNA copies/ml in samples in the acute active phase of B19 infection (DNA+, IgM+, IgG−), 4.3 × 106 B19 DNA copies/ml in samples in the active phase (DNA+, IgM+, IgG+), 3.7 × 105 genome copies/ml in samples in the long‐lasting active phase (DNA+, IgM−, IgG+) with a statistically significant reduction of B19 DNA content between the group of sera in the acute active phase and the group of sera in the active phase of B19 infection. The high levels of sensitivity, specificity, and rapidity provided by the LightCycler technology for the detection and quantitation of B19 DNA represent a significant improvement for the laboratory diagnosis of B19 infection. J. Med. Virol. 67:275–281, 2002. © 2002 Wiley‐Liss, Inc.