Detection of arboviruses
A previously described multiplex reverse transcription (RT)-PCR-HRM test
was initially utilised for the detection of arboviruses within theFlavivirus, Alphavirus, Nairovirus, Phlebovirus, Orthobunyavirus,
and Thogotovirus genera (Villinger et al., 2017) (Supplementary
Table 1). This was preceded by cDNA synthesis using the High Capacity
cDNA Reverse Transcription (RT) kit (Applied Biosystems, Lithuania) in a
20-µl reaction mixture that contained 10 µl nucleic acid extract, 1 U/μl
RNase inhibitor, 100 Mm dNTPs, 1X RT buffer, 2.5 u/µl reverse
transcriptase enzyme and 40 u/µl non-ribosomal random hexa-nucleotide
primers (Endoh et al., 2005). The reactions were performed in a
SimpliAmp thermocycler (Applied Biosystems, Singapore) using previously
described thermal cycling conditions (Ajamma et al., 2018). The 10-µl
reaction mixture for the multiplex PCR-HRM contained 1 µl cDNA template,
5 μl of 2x MyTaq HS Mix (Bioline, UK) and 1 μl of 50 μM SYTO-9 (Life
Technologies, USA). Multiplex PCR-HRM reactions were performed in a
Rotor-Gene Q real-time PCR thermocycler (Qiagen, Hilden, Germany) using
touchdown thermal cycling conditions described in detail elsewhere
(Villinger et al., 2017). Each run included cDNA of the sindbis virus as
a positive control and no-template extraction controls and molecular
grade water as PCR negative controls. HRM profiles were visualised with
Rotor-Gene Q Series software 2.1.0. All positive samples were separately
re-run using primer mixes for each of alphaviruses, flaviviruses, and
nairoviruses and the same conditions for the multiplex PCR-HRM runs
(Villinger et al., 2017) (Supplementary Table 1). Amplicons from
singleplex runs were purified with an Exo 1-rSAP combination (Biolabs,
UK) and submitted for bidirectional sequencing to Macrogen
(Netherlands). Larger fragments using a conventional PCR assay that
targets the Nairovirus L-polymerase gene (Supplementary Table 1)
were also amplified, purified, and sequenced as previously described
(Honig et al., 2004).