For Publishers
DiscoveryMetadataPeer reviewHostingPublishing
For Researchers
JoinPublishReviewCollect
Register
Blog
About
Search
Advanced search
My ScienceOpen
Search
For Publishers
For Researchers
Blog
About
20
views
44
references
 Top references
cited by
28
    
0 reviews
 Review
0
comments
 Comment
0
recommends
+1 Recommend
1 collections
    0
    shares
      15
      similar
       All similar
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      An Economical Tandem Multiplex Real-Time PCR Technique for the Detection of a Comprehensive Range of Respiratory Pathogens

      research-article

      Read this article at

      ScienceOpenPublisherPMC
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          This study used real-time PCR assays to screen small sample volumes for a comprehensive range of 35 respiratory pathogens. Initial thermocycling was limited to 20 cycles to avoid competition for reagents, followed by a secondary real-time multiplex PCR. Supplementary semi-nested human metapneumovirus and picornavirus PCR assays were required to complete the acute respiratory pathogen profile. Potential pathogens were detected in 85 (70%) of pernasal aspirates collected from 121 children with acute respiratory symptoms. Multiple pathogens were detected in 29 (24%) of those samples. The tandem multiplex real-time PCR was an efficient method for the rapid detection of multiple pathogens.

          Related collections

          Most cited references44

          • Record: found
          • Abstract: found
          • Article: not found

          Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

          Christian Drosten, Stephan Günther, Wolfgang Preiser … (2003)
          The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS. Copyright 2003 Massachusetts Medical Society
          Article 0 comments Cited 2346 times – based on 0 reviews     Review now
            • Record: found
            • Abstract: found
            • Article: not found

            A newly discovered human pneumovirus isolated from young children with respiratory tract disease

            Bernadette van den Hoogen, Jan C de Jong, Jan Groen … (2001)
            From 28 young children in the Netherlands, we isolated a paramyxovirus that was identified as a tentative new member of the Metapneumovirus genus based on virological data, sequence homology and gene constellation. Previously, avian pneumovirus was the sole member of this recently assigned genus, hence the provisional name for the newly discovered virus: human metapneumovirus. The clinical symptoms of the children from whom the virus was isolated were similar to those caused by human respiratory syncytial virus infection, ranging from upper respiratory tract disease to severe bronchiolitis and pneumonia. Serological studies showed that by the age of five years, virtually all children in the Netherlands have been exposed to human metapneumovirus and that the virus has been circulating in humans for at least 50 years.
            Article 0 comments Cited 797 times – based on 0 reviews     Review now
              • Record: found
              • Abstract: found
              • Article: not found

              Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia.

              Patrick C. Y. Woo, Susanna Lau, Chung-ming Chu … (2005)
              Despite extensive laboratory investigations in patients with respiratory tract infections, no microbiological cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here we report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-year-old man with pneumonia who had just returned from Shenzhen, China. Quantitative reverse transcription-PCR showed that the amount of CoV-HKU1 RNA was 8.5 to 9.6 x 10(6) copies per ml in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with immunoglobulin M (IgM) titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, respectively. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic analysis reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, negative for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an additional specimen, with a viral load of 1.13 x 10(6) copies per ml, from a 35-year-old woman with pneumonia. Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans.
              Article 0 comments Cited 788 times – based on 0 reviews     Review now
                All references

                Author and article information

                Journal
                Journal ID (nlm-ta): Viruses
                Title: Viruses
                Publisher: Molecular Diversity Preservation International (MDPI)
                ISSN (Electronic): 1999-4915
                Publication date Collection: June 2009
                Publication date (Electronic): 8 June 2009
                Volume: 1
                Issue: 1
                Pages: 42-56
                Affiliations
                [1 ] Department of Microbiology, Pathwest Laboratory Medicine WA, QEII Medical Centre, Nedlands, WA 6009 Australia
                [2 ] School of Biomedical, Biomolecular and Chemical Sciences M502, University of Western Australia, Nedlands, WA 6009 Australia, E-Mails: gerry.harnett@ 123456health.wa.gov.au (G-B.H); gshellam@ 123456cyllene.uwa.edu.au (G-R.S.); david.smith@ 123456health.wa.gov.au (D-W.S)
                Author notes
                [* ]Author to whom correspondence should be addressed; E-mail: glenys.chidlow@ 123456health.wa.gov.au ; Tel.: +61 8 93463260; Fax: +61 8 93463960
                Article
                Publisher ID: viruses-01-00042
                DOI: 10.3390/v1010042
                PMC ID: 3185464
                PubMed ID: 21994537
                SO-VID: 3f67e460-976a-456b-8fde-a535644e787e
                Copyright © © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                Date received : 5 May 2009
                Date revision received : 2 June 2009
                Date accepted : 5 June 2009
                Categories
                Subject: Article

                ScienceOpen disciplines: Microbiology & Virology
                Keywords: mixed infections,tandem multiplex real-time pcr,respiratory pathogens
                Data availability:
                ScienceOpen disciplines: Microbiology & Virology
                Keywords: mixed infections, tandem multiplex real-time pcr, respiratory pathogens

                Comments

                Comment on this article

                Related Documents Log
                scite_

                Similar content15

                See all similar

                Cited by28

                See all cited by

                Most referenced authors1,057

                See all reference authors