使用微孔板发光计开发用于检测和定量细小病毒B19 DNA的化学发光竞争性PCR。

Development of a chemiluminescence competitive PCR for the detection and quantification of parvovirus B19 DNA using a microplate luminometer.

作者信息

Fini F, Gallinella G, Girotti S, Zerbini M, Musiani M

机构信息

Institute of Chemical Science, Division of Microbiology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.

出版信息

Clin Chem. 1999 Sep;45(9):1391-6.

PMID:10471640

Abstract

BACKGROUND

Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19.

METHODS

Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products.

RESULTS

Luminol-based systems displayed constant emission but had a higher detection limit (100-1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera.

CONCLUSIONS

The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.

摘要

背景

病毒核酸的定量聚合酶链反应（PCR）在临床诊断、风险评估及抗病毒治疗监测中可能具有重要作用。我们希望开发一种针对细小病毒B19的化学发光竞争性PCR（cPCR）方法。

方法

细小病毒DNA靶序列和竞争序列共同扩增并直接标记。扩增产物随后分别与特异性生物素标记探针杂交，捕获到包被链霉亲和素的酶联免疫吸附测定（ELISA）微孔板上，并使用过氧化物酶的化学发光底物进行免疫酶法检测。化学发光信号通过微孔板发光计进行定量分析，并与扩增产物的量相关联。

结果

基于鲁米诺的系统显示出恒定的发射，但与基于吖啶的系统（20个基因组拷贝）相比，检测限更高（100 - 1000个基因组拷贝）。化学发光底物的检测限低于比色底物（20个基因组拷贝对比50个基因组拷贝）。在化学发光cPCR中，滴定曲线在100个以上靶基因组拷贝时显示出线性相关性。化学发光cPCR在来自细小病毒感染患者的6份血清样本中呈阳性，在6份对照血清中呈阴性。