Jan 15, 2026

Public workspaceDevelopment and validation of RT-PCR and nested PCR assays for detection of Sabiá virus (SABV)

PLOS Neglected Tropical Diseases
DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2z293l1y/v1
  • Ingra Morales Claro1
  • 1University of Kentucky
Ingra Morales
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2z293l1y/v1
External link: https://doi.org/10.1371/journal.pntd.0014008
Protocol Citation: Ingra Morales Claro 2026. Development and validation of RT-PCR and nested PCR assays for detection of Sabiá virus (SABV). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2z293l1y/v1
Manuscript citation:
Claro IM, Manuli ER, Silva CAMd, Coletti TM, Lemey P, Nastri AC, Casadio LVB, Duarte-Neto AN, Quick J, Romano CM, Whittaker C, Hill SC, Prete CA, Candido DS, Moreira FRR, Ramundo MS, Valença IN, Jesus JGd, Sales FCS, Cunha MS, Guerra JM, Mendes-Correa MC, Tozetto-Mendoza TR, Fumagalli MJ, Ho Y, Simmonds P, Ng WM, Bowden TA, Souza WMd, Pybus OG, Levin AS, Loman N, Sabino EC, Faria NR (2026) Genomic characterization of Sabiá virus in Brazil, 2019–2020: Implications for diagnostics, virus evolution, and receptor binding. PLOS Neglected Tropical Diseases 20(2). doi: 10.1371/journal.pntd.0014008
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 14, 2026
Last Modified: January 15, 2026
Protocol Integer ID: 238667
Keywords: SABV, arenavirus, RT-PCR, Nested PCR , Molecular Biology, Virology, detection of sabiá virus, sabiá virus, sabv, pcr
Abstract
Note
This protocol describes the design, validation, and application of conventional RT-PCR and nested PCR assays for detection of Sabiá virus (SABV). Primer sets were designed based on the 1990 reference genome and newly generated SABV genomes, targeting conserved regions of the nucleoprotein gene. The protocol includes RT-PCR screening and nested PCR amplification optimized for low-viremia samples.
Troubleshooting
Materials
Reagents
Q5 DNA Polymerase (New England Biolabs)
5× Q5 Reaction Buffer
dNTPs (10 mM)
ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs)
Random Hexamers (Thermo Fisher Scientific)
Nuclease-free water
E-Gel EX Agarose 2% (Thermo Fisher Scientific)
E-Gel DNA Sizing Ladder
RNAse P primers and probe (optional internal control)
Equipment
Thermal cycler
Gel electrophoresis system (E-Gel)
UV or blue-light transilluminator
Primer tables
RT-PCR

ABCDE
NameSequence 5’ - 3’Numer of basesAmplicon lenghtTm
Pos_29FGTCACGCTTAAATCTTTGATTGC23350 bp55°C
Pos_381RACAGACACCTCAAGACACCA20  
All_3FTCACGCTTAAATCTTTGATTGCG23121 bp65°C
All_3RGCAGCAATYAAGCTCACTGC20  
 f = forward primer, r = reverse primer, bp = base pairs, Tm = melting temperature.

Nested-cPCR
ABCDE
NameSequence 5’ - 3’Numer of basesAmplicon lenghtTm
Pos_29F_outerGTC ACG CTT AAA TCT TTG ATT GC23350 bp55 oC*
Pos_381R_outerACA GAC ACC TCA AGA CAC CA20 
Pos_171F_innerAAC CTG TGG AAG AGT GGC CT20200 bp52 oC*
Pos_381R_innerACA GAC ACC TCA AGA CAC CA20  
S_outer_1_fTCA GTG CAG GGA CAG ATC CA23494 bp69 oC*
S_outer_1_rTCC  CTG  AGA AGA GGG CTC AG20  
S_inner_1_fTAC AAC CCC TGG AGA CCT CA20112 bp68 oC*
S_inner_1_rTCA GGA GGT GTG TAC CTG GG20  
RNAse P-fAGA TTT GGA CCT GCG AGC G19 65.5 oC
RNAse P-rGAG CGG CTG TCT CCA CAA GT20 67.4 oC
RNAse P-pHEX-TTC TGA CCT /ZEN/ GAA GGC TCT GCG CG22 71.6 oC
f = forward primer, r = reverse primer, p = probe, bp = base pairs, Tm = melting temperature.

Step-by-step - cDNA synthesis
Per reaction, combine and mix thoroughly the following in a 0.2 mL PCR tube:

AB
ComponentVolume
Random Hexamers (50 µM)1 µL
RNA7 µL
Total8 µL
Place on the thermocycler and start the program – 65 ° C for 5 min – when finished, place on ice. Add the following to each tube:

AB
ComponentVolume
Protoscript II Reaction Mix (2X)10 µL
Protoscript II Enzyme Mix (10X)2 µL
Total12 µL
Incubate on thermocycler as follows:
AB
TemperatureTime
25°C5 minutes
48°C15 minutes
80°C5 minutes
HoldHold
Step-by-step - RT-PCR protocol
Combine the following per reaction to create the amplification master mix:
AB
ComponentVolume
Q5 Reaction Buffer 5x5 µL
dNTPs (10 mM)0.5 µL
Q5 DNA Polymerase0.25 µL
Forward primer (10 µM)1.25 µL
Reverse primer (10 µM)1.25 µL
cDNA2.5 µL
Nuclease-free water25 µL
Total25 µL
Incubate on thermocycler as follows:
ABC
TemperatureTimeCycles
98 °C1 minute1
98 °C30 seconds35
Annealing at primer-specific Tm30 seconds35
72 °C1 minute35
72 °C2 minutes1
4 °CHold1

Nested-cPCR protocol
Outer PCR

Combine the following per reaction to create the amplification master mix:

AB
ComponentVolume
Q5 Reaction Buffer 5x5 µL
dNTPs (10 mM)0.5 µL
Q5 DNA Polymerase0.25 µL
Forward primer (10 µM)1 µL
Reverse primer (10 µM)1 µL
cDNA2.5 µL
Nuclease-free water25 µL
Total25 µL
Incubate on thermocycler as follows:

ABC
TemperatureTimeCycles
98 °C1 minute1
98 °C30 seconds35
Annealing at primer-specific Tm30 seconds35
72 °C1 minute35
72 °C2 minutes1
4 °CHold1
Inner PCR

Note
Use 2.5 µL of the outer PCR product as template and repeat the reaction using inner primers under the same cycling conditions.
Visualization

Gel electrophoresis

Note
Load 5 µL of PCR product mixed with 15 µL nuclease-free water on a 2% E-Gel EX agarose gel. Run according to manufacturer’s instructions until bands are clearly visible by transillumination.
Notes
Degenerate bases were incorporated in selected primers to accommodate sequence variability.
Shorter amplicons were prioritized for degraded RNA.
Nested PCR should be handled carefully to avoid cross-contamination.