实时荧光定量 PCR 在急性肝衰竭患者细小病毒 B19 感染鉴别诊断中的应用。

Quantitative real-time PCR for differential diagnostics of parvovirus B19 infection in acute liver failure patients.

机构信息

a Laboratório de Desenvolvimento Tecnológico em Virologia , Instituto Oswaldo cruz, Fundação Oswaldo Cruz , Rio de Janeiro , Brasil.

b Departamento de Microbiologia e Parasitologia , Instituto Biomédico, Universidade Federal Fluminense , Niterói , Brasil.

出版信息

Expert Rev Mol Diagn. 2019 Mar;19(3):259-266. doi: 10.1080/14737159.2019.1582333. Epub 2019 Feb 26.

DOI:10.1080/14737159.2019.1582333

PMID:30773069

Abstract

BACKGROUND

Human Parvovirus B19 (B19V) is a common pathogen worldwide. After primary infection, B19V-DNA may permanently persist in non-erythroid tissues, including the liver of patients with acute liver failure (ALF).

OBJECTIVE

To validate a real-time PCR (qPCR) for the quantification of B19V-DNA, in order to establish a differential diagnosis for B19V infection in ALF patients.

METHODS

The qPCR techniques were based on Sybr Green® and TaqMan® methodologies. To evaluate the quality parameters of both methods, samples from patients with or without B19V infection were tested. The diagnostic utility of qPCR in the detection B19V-DNA in patients with ALF was evaluated by testing archived serum and hepatic tissue explants from 10 patients.

RESULTS

The Sybr Green® methodology showed 97% efficiency, the limits of detection and quantification were 62.6 and 53,200 copies/mL, respectively. The TaqMan® methodology showed 95% efficiency, the limits of detection and quantification were 4.48 and 310 copies/mL, respectively. A false positive result was found only with the Sybr Green® methodology. Among ALF patients without defined etiology, three (30%) were positive for B19V DNA in serum and liver.

CONCLUSION

The qPCR methods validated here were effective in clarifying uncommon cases of B19V-related ALF and are fit for differential diagnosis of ALF causes.

摘要

背景

人类细小病毒 B19（B19V）是一种在全球范围内普遍存在的病原体。初次感染后，B19V-DNA 可能会在非红细胞组织中永久存在，包括急性肝衰竭（ALF）患者的肝脏。

目的

验证用于定量检测 B19V-DNA 的实时 PCR（qPCR），以便为 ALF 患者的 B19V 感染建立鉴别诊断。

方法

qPCR 技术基于 Sybr Green®和 TaqMan®方法学。为了评估这两种方法的质量参数，测试了患有或未患有 B19V 感染的患者样本。通过测试 10 名 ALF 患者的存档血清和肝组织外植体，评估 qPCR 在检测 ALF 患者 B19V-DNA 中的诊断效用。

结果

Sybr Green®方法学的效率为 97%，检测限和定量限分别为 62.6 和 53,200 拷贝/mL。TaqMan®方法学的效率为 95%，检测限和定量限分别为 4.48 和 310 拷贝/mL。仅 Sybr Green®方法学出现假阳性结果。在没有明确病因的 ALF 患者中，有 3 名（30%）在血清和肝脏中 B19V DNA 检测呈阳性。