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

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 x 10(7) to 5 x 10(7)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 x 10(4) to 8.1 x 10(9)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.

SUBMITTER: Choi JH

PROVIDER: S-EPMC7108235 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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

Choi Jang-Hoon JH Chung Yoon-Seok YS Kim Ki-Soon KS Lee Wan-Ji WJ Chung Il Yup IY Oh Hee-Bok HB Kang Chun C

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20080418 3

<h4>Background</h4>Human bocavirus (HBoV) is a parvovirus that has been recently detected in patients with respiratory illness.<h4>Objectives</h4>We developed a sensitive, specific, and quantitative real-time PCR assay based on the TaqMan method for HBoV detection and quantification in respiratory specimens.<h4>Study design</h4>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 acu ...[more]

PMID: 18378492

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Project description:Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R(2) > 0.98) over a 7-log-unit dynamic range down to 10(1) B. atrophaeus cells or spores. Quantification of S. marcescens (R(2) > 0.98) was linear over a 6-log-unit dynamic range down to 10(2) S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

Dataset Information

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

Publications

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

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