Volume 21, Number 1—January 2015
Protocol for Metagenomic Virus Detection in Clinical Specimens1
|Purpose, method and supplier||Score†|
|Virus release/ homogenization|
|Ultrasonic (Sonopuls; Bandelin Electronic, Berlin, Germany)||+2|
|Dounce homogenizer (Kleinfeld Labortechnik, Gehrden, Germany)||+1|
|Qiashredder (QIAGEN, Hilden, Germany)||0|
|Trypsin (Life Technologies, Darmstadt, Germany)||+3|
|FastPrep Homogenizer (MP Biomedicals, Strasbourg, France) (longer homogenization time)||+4|
|Enrichment of virus particles|
|Filtration 0.2- µm filter (Merck-Millipore, Temecula, CA, USA)||+4|
|Filtration 0.45-µm filter (Merck-Millipore)||−2|
|Durapore polyvinylidene fluoride filter tubes (Merck-Millipore)||+2|
|With or without clearing centrifugation||+3|
|Taguchi-optimized centrifugation: 20% sucrose cushion overlaying 80% sucrose cushion and second clearing ultracentrifugation||+4|
|PEG-It virus precipitation (System Biosciences, Mountain View, CA, USA)||+1|
|InRichment Virus Reagent Kit I (Analytik Jena AC, Jena, Germany)||−1|
|Digestion/removal of host nucleotides|
|Turbo DNA-free (Ambion, Darmstadt, Germany) 30 min at 37°C with centrifugation||+4|
|RiboMinus Eukaryote Kit (Invitrogen Life Technologies, Grand Island, NY, USA)||+1|
|QIAamp UltraSens Virus Kit (QIAGEN)||+2|
|PureLink Viral RNA⁄DNA (Invitrogen Life Technologies)||+1|
|QIAamp MinElute Virus Spin Kit (QIAGEN)||−1|
|RTP DNA/RNA Virus Mini Kit (Invitek, Berlin, Germany)||−2|
|RTP DNA/RNA Virus Ultra Sense (Invitek)||0|
|NucleoSpin RNA II (Macherey Nagel, Dueren, Germany)||0|
|NucleoSpin DNA (Macherey Nagel)||+2|
|Phenol chloroform extraction (Carl Roth GmbH, Karlsruhe, Germany)||+3|
|TRIzol LS reagent (Life Technologies)||+4|
|N12 random primer||+3|
|N10 random primer||+2|
|K primer‡ (7)||+3|
|3′ locked random primer(8)||+1|
*WTA, whole transcriptome amplification; WGA, whole genome amplification.
†For every relative quantification result that increased the ratio between host and virus nucleic acids, 1 point was assigned (maximum +4 points if the method led to a better detectability for all 4 viruses). For every decrease, 1 point was subtracted (minimum −4 points).
‡WTA and K primer showed similar results. However, when we considered the lower costs and ease of handling of K primers,
we used K primers for this protocol.
- Fauci AS, Morens DM. The perpetual challenge of infectious diseases. N Engl J Med. 2012;366:454–61.
- Jones KE, Patel NGN, Levy MA, Storeygard A, Balk D, Gittleman JL, Global trends in emerging infectious diseases. Nature. 2008;451:990–3.
- 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.
- 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;441:95–106.
- 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.
- 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;86:8221–31.
- Stang A, Korn K. Characterization of virus isolates by particle-associated nucleic acid PCR. J Clin Microbiol. 2005;43:716–20.
- 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.
- King AM, Adams MJ, Carstens EB, Ninth L. Report of the international committee on taxonomy of viruses. San Diego: Elsevier Academic Press; 2011.
- Tidona CA, Darai G. The Springer index of viruses. Berlin: Springer; 2001.
- 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.
- Taguchi G. Introduction to quality engineering : designing quality into products and processes. Tokyo: Asian Productivity Organization; 1986.
- 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;55:611–22.
- Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.
- 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.
- Schroeder K, Nitsche A. Multicolor, multiplex real-time PCR assay for the detection of human-pathogenic poxviruses. Mol Cell Probes. 2010;24:110–3.
- Kurth A. Possible biohazard risk from infectious tissue and culture cells preserved with RNAlater. Clin Chem. 2007;53:1389–90.
- 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;322:225–30.
- Marston DA, McElhinney LM, Ellis RJ, Horton DL, Wise EL, Leech SL, Next generation sequencing of viral RNA genomes. BMC Genomics. 2013;14:444.
- 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;86:1888–92.
- 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;86:10999–1012.
- 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;86:4620–30.
- 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;84:13004–18.
- 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;84:6955–65.
- 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.
1Preliminary results of this study were presented at the Biodefense and Emerging Infectious Diseases Meeting, January 29, 2014, Washington DC, USA.