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Volume 21, Number 1—January 2015

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 1

Comparison of methods used to develop a protocol for metagenomic virus detection in infectious disease settings*

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
Fractionated filtration −1
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
Nucleotide extraction
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
WTA‡ +3
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.

Main Article

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

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