Table 1.

Real-time PCR arbovirus assay design

Virus	Primer identifier	Primer function	Primer sequence	Product size
BFV*	BFV-27	Forward	5′-CCTCAGGGCCACTCACCATA-3′	85 bp
	BFV-111	Reverse	5′-TTCTGCTAGTGTCTTGCACATACTACA-3′
	BFV-49	Probe	5′-6FAM-ACAACAACGACTATAATAGCTACCACA-BHQ1-3′
KUNV†	KUN-53	Forward	5′-CATTGGTTGAGGATACAGTATTG-3′	132 bp
	KUN-184	Reverse	5′-CCTGACTTCCTCACTAAAATTTT-3′
	KUN-105	Probe	5′-VICAAGCTAAACTTCTACACATAATAACA-MGBNFQ-3′
MVEV‡	MVE-503	Forward	5′-AGCCGAAGCGGTCATAGGT-3′	72 bp
	MVE-574	Reverse	5′-CATGTGCGGACTGCAAATTT-3′
	MVE-527	Probe	5′-6FAM-TTTCAATGCTTTCAATGTCA-MGBNFQ-3′
RRV§	RRV-9	Forward	5′-CGATGACGTGGGTACAGAGGAT-3′	79 bp
	RRV-87	Reverse	5′- GTTACCAAGACCAGCACAACCA-3′
	RRV-33	Probe	5′-6FAM-TAGAGGGCCAGCCCACCTAACCCACTG-BHQ1-3′

BFV = Barmah Forest virus; bp = base pair; KUNV = Kunjin virus; MVEV = Murray Valley encephalitis virus; PCR = polymerase chain reaction; RRV = Ross River virus.

^*BFV target was the E1 region -3′ UTR junction of the structural polyprotein gene (accession U73745; 10,967–11,051 nt).

^†KUNV target was the NS5 region -3′ UTR junction of the non-structural polyprotein gene (accession D00246.1; 10,352–10,483 nt).

^‡MVEV target was the NS5 region of the nonstructural polyprotein gene (accession AF161266.1; 9,563–9,634 nt).

^§RRV target was the E1 region of the structural polyprotein gene (accession GQ433359; 11,234–11,312 nt).