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Protocol for metagenomic virus detection in clinical specimens.

Kohl C, Brinkmann A, Dabrowski PW, Radonić A, Nitsche A, Kurth A - Emerging Infect. Dis. (2015)

Bottom Line: Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population.The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing.We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches.

View Article: PubMed Central - PubMed

ABSTRACT
Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health. Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population. We quantifiably compared classical and modern approaches of virus purification and enrichment in theory and experiments. Eventually, we established an unbiased protocol for detection of known and novel emerging viruses from organ tissues (tissue-based universal virus detection for viral metagenomics [TUViD-VM]). The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches. This TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source.

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Comparison of primers and random amplification methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ (2 – ΔΔCt); (ΔΔCt = Δ purified – Δ unprocessed). Lower panel left y-axis indicates signal-to-noise ratio (RQ) for all viruses tested. The method with the highest score was used to establish the protocol and is shaded in yellow. Red stars indicate highest scores. Diagonally striped area indicates not significant. Ct, cycle threshold. Numbers along baseline indicate method used. 1, control; 2, K primer; 3, 3′ locked primer; 4, N12 primer; 5, N primer; 6, whole transcriptome amplification (QIAGEN, Hilden, Germany); 7, whole genome amplification (QIAGEN).
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Figure 7: Comparison of primers and random amplification methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ (2 – ΔΔCt); (ΔΔCt = Δ purified – Δ unprocessed). Lower panel left y-axis indicates signal-to-noise ratio (RQ) for all viruses tested. The method with the highest score was used to establish the protocol and is shaded in yellow. Red stars indicate highest scores. Diagonally striped area indicates not significant. Ct, cycle threshold. Numbers along baseline indicate method used. 1, control; 2, K primer; 3, 3′ locked primer; 4, N12 primer; 5, N primer; 6, whole transcriptome amplification (QIAGEN, Hilden, Germany); 7, whole genome amplification (QIAGEN).

Mentions: Every step of the TUViD protocol (homogenization of tissue, filtration, digestion, enrichment, extraction, and random amplification) was compared with alternative approaches. Results are shown in Figures 3,4,5,6,7. Each approach was tested with individual samples, which were measured by using 5 PCRs specific for viruses used and host background in 2 replicates (10 reactions/sample): Results were quantified and evaluated in qPCRs for the 4 viruses and presence of host nucleic acids (Technical Appendix; Table 4; Figures 3,4,5,6,7). A scoring system was developed to assess the optimal combination of all 4 viruses (Table 1; Figures 3,4,5,6,7). A preliminary protocol was further validated and adjusted until no host nucleic acids were detectable by qPCR. This protocol maximized the amount of amplified virus nucleic acids. Subsequently, we established an unbiased protocol for the detection of known and novel viruses in infected organ tissues (TUViD-VM).


Protocol for metagenomic virus detection in clinical specimens.

Kohl C, Brinkmann A, Dabrowski PW, Radonić A, Nitsche A, Kurth A - Emerging Infect. Dis. (2015)

Comparison of primers and random amplification methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ (2 – ΔΔCt); (ΔΔCt = Δ purified – Δ unprocessed). Lower panel left y-axis indicates signal-to-noise ratio (RQ) for all viruses tested. The method with the highest score was used to establish the protocol and is shaded in yellow. Red stars indicate highest scores. Diagonally striped area indicates not significant. Ct, cycle threshold. Numbers along baseline indicate method used. 1, control; 2, K primer; 3, 3′ locked primer; 4, N12 primer; 5, N primer; 6, whole transcriptome amplification (QIAGEN, Hilden, Germany); 7, whole genome amplification (QIAGEN).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4285256&req=5

Figure 7: Comparison of primers and random amplification methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ (2 – ΔΔCt); (ΔΔCt = Δ purified – Δ unprocessed). Lower panel left y-axis indicates signal-to-noise ratio (RQ) for all viruses tested. The method with the highest score was used to establish the protocol and is shaded in yellow. Red stars indicate highest scores. Diagonally striped area indicates not significant. Ct, cycle threshold. Numbers along baseline indicate method used. 1, control; 2, K primer; 3, 3′ locked primer; 4, N12 primer; 5, N primer; 6, whole transcriptome amplification (QIAGEN, Hilden, Germany); 7, whole genome amplification (QIAGEN).
Mentions: Every step of the TUViD protocol (homogenization of tissue, filtration, digestion, enrichment, extraction, and random amplification) was compared with alternative approaches. Results are shown in Figures 3,4,5,6,7. Each approach was tested with individual samples, which were measured by using 5 PCRs specific for viruses used and host background in 2 replicates (10 reactions/sample): Results were quantified and evaluated in qPCRs for the 4 viruses and presence of host nucleic acids (Technical Appendix; Table 4; Figures 3,4,5,6,7). A scoring system was developed to assess the optimal combination of all 4 viruses (Table 1; Figures 3,4,5,6,7). A preliminary protocol was further validated and adjusted until no host nucleic acids were detectable by qPCR. This protocol maximized the amount of amplified virus nucleic acids. Subsequently, we established an unbiased protocol for the detection of known and novel viruses in infected organ tissues (TUViD-VM).

Bottom Line: Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population.The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing.We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches.

View Article: PubMed Central - PubMed

ABSTRACT
Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health. Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population. We quantifiably compared classical and modern approaches of virus purification and enrichment in theory and experiments. Eventually, we established an unbiased protocol for detection of known and novel emerging viruses from organ tissues (tissue-based universal virus detection for viral metagenomics [TUViD-VM]). The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches. This TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source.

Show MeSH
Related in: MedlinePlus