Technologies for Arbovirus Detection and Surveillance

CADDE is working on the development, validation and application of novel genome sequencing technologies to characterise known and unknown arboviruses in human populations, in sentinel vector (mosquito) populations, and in non-human reservoirs.

While whole genome sequencing provides powerful information to understand the spread and dynamics of an outbreak in real-time, traditional virologic methods (i.e. detection of virus genome through RT-qPCR or isolation of the virus in cell lines) and/or serological methods such as ELISA or PRNT remain the gold standard for arbovirus detection and surveillance across the Americas. The state-of-the-art sequencing approaches should build upon existing tools and complement standard surveillance methods. Detailed information on detection and laboratory diagnosis can be found in the Pan American Health Organization website for several arboviruses, including yellow fever virus, dengue virus, Zika virus, chikungunya virus and mayaro virus.

Multiplex-PCR

The multiplex-PCR protocol was developed by Josh Quick and Nick Loman as part of the ZiBRA project and can be found here.

Primer Schemes

Each scheme is a set of oligonucleotide primer pairs that generate overlapping products. Validated and unvalidated primer schemes (see Data Availability Disclaimer below) published in the citations below can be downloaded in CSV format:

Data Availability Disclaimer

Primer schemes reported here is being shared pre-publication to help the research and public health communities respond to ongoing or future arbovirus disease outbreaks. Please note though that this data is still based on work in progress and should be considered preliminary. Our analysis and validation of unpublished primer schemes is ongoing and publications communicating our findings are in preparation. CADDE will distribute these data to any entity or collaborator that needs it. If you intend to use these data prior to our publication, please contact Profs. Ester Sabino, Nuno Faria, and Nick Loman to coordinate.

SmartPlex Metagenomics

This is a Smart-Seq (Switch Mechanism at the 5′ End of RNA Templates) protocol using random 9-n priming. The protocol is compatible with the Oxford Nanopore Technologies RLB rapid, barcoded adapters available from the SQK-RPB004 kit. This protocol has been developed and tested by Josh Quick and Ingra Claro. Please cite contact Josh Quick and Nick Loman for additional information and appropriate citation.

List of reagents:

  • Ultrafree-MC Centrifugal Filter (10228490)
  • ZymoBIOMICS DNA Microprep Kit with Lysis Tubes (D4301)
  • Quick-RNA Viral Kit (R1034)
  • Turbo DNase (Thermo Fisher, AM2238)
  • Clean up kit Zymo Research, (Zymo, R1015)
  • SuperScript IV (15307696)
  • RNase OUT (10777019)
  • Ampure XP 60 ml (A63881)
  • LongAmp Taq 2X Master Mix (M0287)

List of oligos required:

  • RLB TSO (RNA oligo)
  • GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATrGrGrG
  • RLB RT 9N
  • TTTTTCGTGCGCCGCTTCAACNNNNNNNNN
  • RLB PCR (not required but useful for testing)
  • TTTTTCGTGCGCCGCTTCA

Step 1. Centrifugal filtration

  • Transfer up to 500 ul directly onto a Ultrafree-MC Centrifugal Filter column
  • Spin at 5,000 xg for 1 min
  • Recover filtrate into 1.5 ml tube
  • Remove basket and discard
  • Close lid and place on ice

Step 2. Viral RNA extraction

  • In a 2 ml Eppendorf tube combine 200 ul sample, 200 ul DNA/RNA Shield (2x concentrate) and mix well by pipetting
  • Add 800 ul Viral RNA Buffer and mix well by pipetting
  • Load 600 ul onto a column in a collection tube and spin at 10,000 xg for 15 secs discard flow through, place in a new collection tube
  • Add 500 ul Viral wash buffer and spin at 10,000 xg for 15 sec, discard flow through
  • Add 500 ul 100% ethanol and spin at 10,000 xg for 1 min, discard flow through, place in a clean 1.5 ml tube
  • Add 15 ul of DNA/RNA-Free Water and incubate at RT for 3 minutes
  • Spin at 10,000 xg for 15 sec

Step 3. DNase treatment

  • Set heat block to 37°C
  • Set up the following reaction
  • RNA – 44 ul
  • 10X TURBO DNase Buffer 5 ul
  • TURBO DNase –  1 ul
  • Incubate at 37°C for 30 minutes

Step 4. DNase cleanup

  • Add 100 ul RNA Binding Buffer and mix by vortexing 5 secs and spin down
  • Add 150 ul 100% ethanol and mix by vortexing for 15 secs and spin down
  • Transfer 300 ul to a Zymo-Spin IC column in a 2 ml collection tube and spin at 6000 xg for 15 secs discard flow through
  • Add 400 ul RNA Prep Buffer and spin at 6000 xg for 15 sec, discard flow through
  • Add 700 ul RNA Wash Buffer and spin at 6000 xg for 15 sec, discard flow through and place in a new 1.5 ml tube
  • Add 10 ul DNase/RNase Free Water and incubate at RT for 1 min
  • Spin at 6000 xg for 15 secs
  • Label as ‘Viral RNA’ and place on ice

Step 5. Viral DNA extraction

  • Set heat block to 55°C
  • In a 2 ml Eppendorf tube combine 200 ul sample, 200 ul DNA/RNA Shield (2X concentrate), 20 ul Proteinase K and mix well by pipetting
  • Incubate at 55°C for 30 minutes
  • Set heat block to 60°C
  • Add 1,200 ul binding buffer and mix well
  • Load 800 ul onto a Zymo-Spin IIC-Z column in a collection tube and spin at 8,000 xg for 15 secs, discard the flow-through and reload as many times as required
  • Transfer to a new collection tube add 400 ul DNA Wash Buffer 1 and spin at 8,000 xg for 15 secs, discard flow-through
  • Add 700 ul DNA Wash Buffer 2 and spin at 8,000 xg for 15 secs
  • Add 200 ul DNA Wash Buffer 2 and spin at 8,000 xg for 1 min
  • Transfer to a new 1.5 ml tube and 50 ul DNA/RNA Free Water preheated to 60°C to the column, incubate at RT for 1 min
  • Spin at 8,000 xg for 1 min

Step 6. Smart-9n amplification

  • Combine the following:
  • RLB RT 9N (2 uM) 1 ul
  • dNTPs (10 mM ea.) 1 ul
  • Template RNA 10 ul
  • Mix and spin down
  • Incubate at 65°C for 5 mins then cool on ice
  • Make up the following master mix
  • SSIV buffer (5x) 4 ul
  • DTT (100 mM) 1 ul
  • RNase OUT 1 ul
  • SS IV RTase (200 U/ul) 1 ul
  • RLB TSO (2 uM) 1 ul
  • Add 8 ul to 12 ul annealed RNA

Start the following program

  • 42°C for 90 mins
  • 70°C for 10 mins

Amplify by adding 5 ul cDNA to each PCR reaction:

  • LongAmp Taq 2X master mix 25 ul
  • RLB (01-12) 0.5 ul
  • NFW – 19.5 ul

Start the following PCR program:

  • 95°C 45 secs
  • 26 cycles 95°C 15 secs
  • 56°C 15 secs
  • 65°C 5 mins
  • 65°C 10 mins
  • 4°C hold
  • Clean up products with 1x Ampure XP and elute in 30 ul EB

Step 7. Quality control

  • Quantify DNA using Qubit HS
  • Run on genomic DNA TapeScreen

Step 8. Adapter attachment

  • Pool all barcoded products to a total of 200 fmol in 10 μl of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
  • Add 1 ul RAP and mix by pipetting, incubate at RT 5 mins

Protocols availability

The protocols reported here are being shared pre-publication to help the research and public health communities respond to ongoing or future arbovirus disease outbreaks. Please note though that this protocol is still based on work in progress and should be considered preliminary. Additional validation is ongoing and publications communicating our findings are in preparation. CADDE will distribute these data to any entity or collaborator that needs it. If you intend to use this protocol prior to our publication, please contact Profs. Ester Sabino, Nuno Faria, and Nick Loman to coordinate.