Anchor-Based Fluorescent Amplicon Generation Assays (FLAG) for Real-Time Measurement of Human Cytomegalovirus, Epstein-Barr Virus, and Varicella-Zoster Virus Viral Loads

被引：2

作者：

Di Nicola, Alessandro ^{[1
]}

Ghezzi, Elisa ^{[1
]}

Gillio, Federico ^{[1
]}

Zerilli, Francesco ^{[2
]}

Shehi, Erlet ^{[1
]}

Maritano, Diego ^{[1
]}

Panizzo, Massimo ^{[1
]}

Bonelli, Fabrizio ^{[1
]}

Adlerstein, Daniel ^{[1
]}

机构：

[1] Diasorin SpA, I-20014 Nerviano, MI, Italy

[2] Univ Milano Bicocca, Milan, Italy

来源：

CLINICAL CHEMISTRY | 2008年 / 54卷 / 11期

关键词：

D O I：

10.1373/clinchem.2008.106542

中图分类号：

R446 [实验室诊断]; R-33 [实验医学、医学实验];

学科分类号：

1001 ;

摘要：

BACKGROUND: Monitoring the human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), or varicella-zoster virus (VZV) viral load is an important factor in the management of immunosuppressed patients, such as recipients of solid-organ or bone marrow transplants. The advent of real-time PCR technologies has prompted the widespread development of quantitative PCR assays for the detection of viral loads and other diagnostic purposes. METHODS: The fluorescent amplicon generation (FLAG) technology uses the PspG1 restriction enzyme to monitor PCR product generation. We modified the FLAG technology by introducing all accessory oligo-nucleotide "anchor" that stabilizes the binding of the forward primer to the target sequence (a-FLAG). We developed assays for HCMV, EBV, and VZV that incorporated all internal amplification-control reaction to validate negative results and extensively analyzed the performance of the HCMV a-FLAG assay. RESULTS: The 3 assays performed similarly with respect to reaction efficiency and linear range. Compared with a commercially available kit, the HCMV a-FLAG assay results showed good correlation with calculated concentrations (r = 0.9617), excellent diagnostic sensitivity and specificity (99% and 95%, respectively), and similar values for the linear range (1-10(7) copies/mu L), analytical, sensitivity (0.420 copies/mu L), and intra- and interassay imprecision. CONCLUSIONS: The a-FLAG assay is all alternative real-time PCR technology suitable for detecting and quantifying target-DNA sequences. For clinical applications such as the measurement of viral load, a-FLAG assays provide multiplex capability, internal amplification control, and high diagnostic sensitivity and specificity. (C) 2008 American Association for Clinical Chemstry

引用

页码：1900 / 1907

页数：8

共 18 条

[1] FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations [J].