Manaresi Elisabetta, Gallinella Giorgio, Zuffi Elisa, Bonvicini Francesca, Zerbini Marialuisa, Musiani Monica
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Italy.
J Med Virol. 2002 Jun;67(2):275-81. doi: 10.1002/jmv.2218.
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 x 10(1) genome copies/reaction and to detect as few as 3 x 10(0) 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 x 10(9) B19 DNA copies/ml in samples in the acute active phase of B19 infection (DNA+, IgM+, IgG-), 4.3 x 10(6) B19 DNA copies/ml in samples in the active phase (DNA+, IgM+, IgG+), 3.7 x 10(5) 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.
开发了一种实时PCR检测方法，用于临床血清样本中B19 DNA的定量检测。该检测使用LightCycler仪器进行，产物形成通过荧光双链DNA结合染料SYBR Green I持续监测。采用优化的PCR方案，该系统能够将目标DNA定量至低至3×10(1)个基因组拷贝/反应，并能检测低至3×10(0)个基因组拷贝/反应。实时PCR用于检测108份临床怀疑B19感染患者的血清样本中的B19 DNA，与被视为标准的标准化PCR-ELISA相比，显示出92.7%的灵敏度和100%的特异性。使用LightCycler检测，临床血清样本中B19 DNA的检测和定量的整个过程耗时长达90分钟，比PCR-ELISA快五倍。通过实时PCR对阳性样本中的B19 DNA进行定量分析，结果显示，在B19感染的急性活动期样本（DNA阳性、IgM阳性、IgG阴性）中，B19 DNA拷贝数平均为1.1×10(9)/ml，在活动期样本（DNA阳性、IgM阳性、IgG阳性）中为4.3×10(6) B19 DNA拷贝/ml，在持久活动期样本（DNA阳性、IgM阴性、IgG阳性）中为3.7×10(5)个基因组拷贝/ml，B19感染急性活动期血清组与活动期血清组之间的B19 DNA含量有统计学意义的降低。LightCycler技术在B19 DNA检测和定量方面提供的高灵敏度、特异性和快速性，代表了B19感染实验室诊断的显著改进。
