Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 21, Number 1—January 2015
Research

Protocol for Metagenomic Virus Detection in Clinical Specimens1

Claudia KohlComments to Author , Annika Brinkmann, Piotr W. Dabrowski, Aleksandar Radonić, Andreas Nitsche, and Andreas Kurth
Author affiliations: Robert Koch Institute, Berlin, Germany

Main Article

Table 4

PCRs used to develop a protocol for metagenomic virus detection in infectious disease settings*

Virus or DNA Characteristic Primer Gene, sequence (5′→3′), and reference
Reovirus PCR target T3/Germany/Bat/342/08 polymerase gene
Forward primer BatReoF CACCATgTCAAgCTgCTCCC
Reverse primer BatReoR ACCgCCATgTATgTCCTCCAg
Probe Bat Reo FAM-CCCAgTCgCggTCATTACCACTCCg-BBQ
Reference Kohl et al. (11)
Influenza virus PCR target Generic influenza A virus detection
Forward primer M+ 25 AgATgAgTCTTCTAACCgAggTCg
Reverse primer M-124BB CCW-gCAAARACATCYTCAAgTYTCTg
Probe MGB M+64 FAM-TCAggCCCCCTCAA-MGB
Reference Schulze et al. (15)
Vaccinia virus PCR target Generic orthopoxvirus detection
Forward primer OPV F TATTACTTCgATTGCTCATCCAgg
Reverse primer OPV R ACTTCTCACAAATggATTTGAAAATC
Probe OPV TMGB FAM-TCCTTTACgTGATAAATCAT-MGB
Reference Schroeder and Nitsche (16)
Sendai virus PCR target Large polymerase gene
Forward primer Sendai F1 gAGCCGCCTggTCTCCTCAgA
Reverse primer Sendai R1 AAggCATggCggACAgTggC
Probe Sendai MGB FAM-CCgTAgACAATTACACAAgT-MGB
Reference In house design
Chicken DNA PCR target galTBP gene NM_205103.1
Forward primer galTBP F gTgTCCACggTgAATCTTgg
Reverse primer galTBP R TgCATTCTAACATACTTCTCTTACCTTg
Probe galTBP TM 6-FAM-CgTgCCCGAAATgCTgAATATAATCCCA-BBQ
Reference Kurth (17)
Mammalian DNA PCR target c-myc oncogene HUMMYCF03
Forward primer C-myc F gCCAgAggAggAACgAgCT
Reverse primer C-Myc R gggCCTTTTCATTgTTTTCCA
Probe C-Myc TM Cy5-ATgCCCTgCgTgACCAgATCC-NFQ
Reference Schroeder and Nitsche (16)

*–, not applicable.

Main Article

References
  1. Fauci  AS, Morens  DM. The perpetual challenge of infectious diseases. N Engl J Med. 2012;366:45461. DOIPubMed
  2. Jones  KE, Patel  NGN, Levy  MA, Storeygard  A, Balk  D, Gittleman  JL, Global trends in emerging infectious diseases. Nature. 2008;451:9903. DOIPubMed
  3. He  B, Li  Z, Yang  F, Zheng  J, Feng  Y, Guo  H, Virome profiling of bats from Myanmar by metagenomic analysis of tissue samples reveals more novel mammalian viruses. PLoS ONE. 2013;8:e61950. DOIPubMed
  4. Baker  KS, Leggett  RM, Bexfield  NH, Alston  M, Daly  G, Todd  S, Metagenomic study of the viruses of African straw-colored fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus. Virology. 2013;441:95106. DOIPubMed
  5. Nakamura  S, Yang  C-S, Sakon  N, Ueda  M, Tougan  T, Yamashita  A, Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach. PLoS ONE. 2009;4:e4219. DOIPubMed
  6. Whon  TW, Kim  M-S, Roh  SW, Shin  N-R, Lee  H-W, Bae  J-W. Metagenomic characterization of airborne viral DNA diversity in the near-surface atmosphere. J Virol. 2012;86:822131. DOIPubMed
  7. Stang  A, Korn  K. Characterization of virus isolates by particle-associated nucleic acid PCR. J Clin Microbiol. 2005;43:71620. DOIPubMed
  8. Clem  AL, Sims  J, Telang  S, Eaton  JW, Chesney  J. Virus detection and identification using random multiplex (RT)-PCR with 3′-locked random primers. Virol J. 2007;4:65. DOIPubMed
  9. King  AM, Adams  MJ, Carstens  EB, Ninth  L. Report of the international committee on taxonomy of viruses. San Diego: Elsevier Academic Press; 2011.
  10. Tidona  CA, Darai  G. The Springer index of viruses. Berlin: Springer; 2001.
  11. Kohl  C, Lesnik  R, Brinkmann  A, Ebinger  A, Radonić  A, Nitsche  A, Isolation and characterization of three mammalian orthoreoviruses from European bats. PLoS ONE. 2012;7:e43106. DOIPubMed
  12. Taguchi  G. Introduction to quality engineering : designing quality into products and processes. Tokyo: Asian Productivity Organization; 1986.
  13. Bustin  SA, Benes  V, Garson  JA, Hellemans  J, Huggett  J, Kubista  M, The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:61122. DOIPubMed
  14. Langmead  B, Salzberg  SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:3579. DOIPubMed
  15. Schulze  M, Nitsche  A, Schweiger  B, Biere  B. Diagnostic approach for the differentiation of the pandemic influenza A(H1N1)v virus from recent human influenza viruses by real-time PCR. PLoS ONE. 2010;5:e9966. DOIPubMed
  16. Schroeder  K, Nitsche  A. Multicolor, multiplex real-time PCR assay for the detection of human-pathogenic poxviruses. Mol Cell Probes. 2010;24:1103. DOIPubMed
  17. Kurth  A. Possible biohazard risk from infectious tissue and culture cells preserved with RNAlater. Clin Chem. 2007;53:138990. DOIPubMed
  18. Schipper  J, Chanson  JS, Chiozza  F, Cox  NA, Hoffmann  M, Katariya  V, The status of the world’s land and marine mammals: diversity, threat, and knowledge. Science. 2008;322:22530. DOIPubMed
  19. Marston  DA, McElhinney  LM, Ellis  RJ, Horton  DL, Wise  EL, Leech  SL, Next generation sequencing of viral RNA genomes. BMC Genomics. 2013;14:444. DOIPubMed
  20. Kohl  C, Vidovszky  MZ, Mühldorfer  K, Dabrowski  PW, Radonić  A, Nitsche  A, Genome analysis of bat adenovirus 2: indications of interspecies transmission. J Virol. 2012;86:188892. DOIPubMed
  21. Wu  Z, Ren  X, Yang  L, Hu  Y, Yang  J, He  G, Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces. J Virol. 2012;86:109991012. DOIPubMed
  22. Ge  X, Li  Y, Yang  X, Zhang  H, Zhou  P, Zhang  Y, Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China. J Virol. 2012;86:462030. DOIPubMed
  23. Donaldson  EF, Haskew  ANA, Gates  JE, Huynh  J, Moore  CJ, Frieman  MB. Metagenomic analysis of the viromes of three North American bat species: viral diversity among different bat species that share a common habitat. J Virol. 2010;84:1300418. DOIPubMed
  24. Li  L, Victoria  JG, Wang  C, Jones  M, Fellers  GM, Kunz  TH, Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses. J Virol. 2010;84:695565. DOIPubMed
  25. Daly  GM, Bexfield  N, Heaney  J, Stubbs  S, Mayer  AP, Palser  A, A viral discovery methodology for clinical biopsy samples utilizing massively parallel next generation sequencing. PLoS ONE. 2011;6:e28879. DOIPubMed

Main Article

1Preliminary results of this study were presented at the Biodefense and Emerging Infectious Diseases Meeting, January 29, 2014, Washington DC, USA.

Page created: January 05, 2015
Page updated: January 05, 2015
Page reviewed: January 05, 2015
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
file_external