Development of real-time PCR assays for detection and quantification of human bocavirus

https://doi.org/10.1016/j.jcv.2008.02.010Get rights and content

Abstract

Background

Human bocavirus (HBoV) is a parvovirus that has been recently detected in patients with respiratory illness.

Objectives

We developed a sensitive, specific, and quantitative real-time PCR assay based on the TaqMan method for HBoV detection and quantification in respiratory specimens.

Study design

Three individual real-time PCR assays were designed to amplify HBoV NS1, NP-1, and VP1 genes. For clinical evaluation, 506 nasal aspirates obtained from patients with acute respiratory tract infections during December 2006 to May 2007 were tested.

Results

Each assay had a broad dynamic range (50 × 107 to 5 × 107 copies of plasmid DNA) and high inter- and intra-assay reproducibility. The detection limit of each assay was 10 genome copies per reaction, and no crossreactivity with other major respiratory viruses or bacteria was detected. Clinical evaluation revealed that 11 (2.1%) of 506 patients diagnosed with upper respiratory tract infections, pneumonia, bronchitis, pharyngitis, or sinusitis had HBoV detected by all three assays, with viral loads ranging from 8.2 × 104 to 8.1 × 109 copies/ml of specimen.

Conclusions

The three assays for HBoV diagnosis and quantification are highly sensitive, specific real-time tools for the reliable epidemiological and pathogenetic study of HBoV infection.

Abbreviations

HBoV
human bocavirus
LRTIs
lower respiratory track infections
PCR
polymerase chain reaction
MGB
minor groove binder
FAM
6-carboxyfluorescein
UNG
uracil-N-glycosylase
ORFs
open reading frames
PIV
parainfluenza virus
RSV
respiratory syncytial virus

Keywords

Human bocavirus
Respiratory diseases
Real-time PCR
Quantification

Cited by (0)

1

These two authors contributed equally to this work.

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