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. 2010 May;82(5):954–960. doi: 10.4269/ajtmh.2010.09-0636

Table 1.

Real-time PCR assay primers, probes, reaction concentrations and sensitivities*

RNA viruses Target Genome accession # Amplicon size Primers/probe Sequence (5′ - 3′) Final conc (µM) Sensitivity (PFU/PCR)†
Ebola Zaire-MGB NP AF086833 76 F565 5′ - TCT GAC ATG GAT TAC CAC AAG ATC - 3′ 0.9 0.001
R640 5′ - GGA TGA CTC TTT GCC GAA CAA TC - 3′ 0.9
p597S 6FAM - AGG TCT GTC CGT TCA A - MGBNFQ 0.2
Ebola Sudan-MGB NP AY729654 80 F1051 5′ - CAT GCA GAA CAA GGG CTC ATT C - 3′ 1.0 0.1
R1130 5′ - CTC ATC AAA CGG AAG ATC ACC ATC - 3′ 1.0
p1079S 6FAM - CAA CTT CCT GGC AAT - MGBNFQ 0.2
Ebola Reston-MGB GP AB050936 55 F1129 5′ - TCA CCG CGA ACC CAA TG - 3′ 0.8 1.0 (34 copies)‡
R1183 5′ - TCG CTT GTC ATG GTT GGA CTT - 3′ 0.8
p1149S 6FAM - ACC ACC ATT GCC C- MGBNFQ 0.2
Ebola Ivory Coast-MGB GP U28006 64 F1123 5′ - CCC ATC TCC GCC CAC AA - 3′ 0.7 1.0 (586 copies)‡
R1186 5′ - GAG TGG AAT CCT CTG AAA CCA ATT - 3′ 0.7
p1143S 6FAM - CGC AGG CGA AGA C - MGBNFQ 0.2
Ebola Zaire-TM GP AF086833 80 F2000 5′ - TTT TCA ATC CTC AAC CGT AAG GC - 3′ 1.0 0.0001 (584 copies)‡
R2079 5′ - CAG TCC GGT CCC AGA ATG TG - 3′ 1.0
p2058A 6FAM - CAT GTG CCG CCC CAT CGC TGC - TAMRA - 3′ 0.1
Ebola Sudan-TM GP AY729654 77 F583 5′ - AGG ATG GAG CTT TCT TCC TCT ATG - 3′ 0.8 0.1
R659 5′ - TAC CCC CTC AGC AAA ATT GAC T - 3′ 0.8
p608SB 6FAM - CAG GCT GGC TTC AAC TGT AAT TTA CAG AGG - TAMRA -3′ 0.1
Ebola Reston-TM VP40 AF522874 80 F5645 5′ - CTA TGG TTA TCA CCC AGG ATT GTG - 3′ 0.9 1.0
R5724 5′ - GTA ACT ATC CTG CTT GTC CAT GTG - 3′ 0.9
p5674S 6FAM - TGC CAC TCT CCA GCC AGC CAT CCG - TAMRA - 3′ 0.1
Ebola Ivory Coast-TM GP U28006 79 F564 5′ - TGT ACA CAA AGT CTC AGG AAC TGG - 3′ 1.0 0.1
R642 5′ - GTC ATA CAG GAA GAA GGC TCC TTC - 3′ 1.0
p589S 6FAM - CCA TGC CCA GGA GGA CTC GCC TTT - TAMRA -3′ 0.1
Ebola Bundibugyo (MGB and TM) NP FJ217161 74 F2016 5′ - ATG GAA ACC AAG GCG AAA CTG - 3′ 0.9 10−6 (RNA dilution)§ (MGB and TM)
R2089 5′ - TAC TTG TGG CAT TGG CTT GTC T - 3′ 0.9
p2045S 6FAM - CGG GTA GCC CCC AAC - MGBNFQ 0.2
or
p2038S 6FAM - ATC CCC ACG GGT AGC CCC CA - TAMRA 0.1
pan-Marburg-MGB assay: (Ravn, Ci67, Musoke, and Angola-MGB) GP EF446131 (Ravn) EF446132 (Ci67) DQ217792 (Musoke)DQ447660 (Angola) 64 F6121 5′ - GAT TCC CCT TTG GAA GCA TCT - 3′ 1.0 0.1 (Ravn) 1.0 (Ci67) 10 (Musoke) 1.0 (Angola)
F6121-1 5′ - GAT TCC CCT TTA GAG GCA TCC - 3′ 1.5
R6184 5′ - CAA CGT TCT TGG GAG GAA CAC - 3′ 1.0
p6144 6′FAM - ACG ATG GGC TTT CAG - MGBNFQ - 3′ 0.2
pan-Marburg-TM assay: (Ravn, Ci67, Musoke, and Angola-TM) GP EF446131 (Ravn)EF446132 (Ci67)DQ217792 (Musoke)DQ447660 (Angola) 64 F6121 5′ - GAT TCC CCT TTG GAA GCA TCT - 3′ 0.5 0.1 (Ravn)10 (Ci67)1.0 Musoke)10 (Angola)
F6121-1 5′ - GAT TCC CCT TTA GAG GCA TCC - 3′ 1.0
R6184 5′ - CAA CGT TCT TGG GAG GAA CAC - 3′ 1.0
p6145A-5 6FAM - AAA CGA TGG GCC TTC AGG GCA GG-TAMRA-3′ 0.1
p6145A-7 6FAM - AAG CGA TGG GCT TTC AGG ACA GG-TAMRA-3′ 0.1
Marburg Musoke-MGB NP DQ217792 65 F391 5′ - CAA CCC GCT TTC TGG ATG TG - 3′ 1.0 10
R455-3 5′ - CTT AAG GGC TAG AAT TAA AGG GCT - 3′ 1.0
p429A 6FAM - TAA TGA GGT TCG TTA GGA A - MGBNFQ 0.2
Marburg Ci67-MGB NP EF446132 65 F391 5′ - CAA CCC GCT TTC TGG ATG TG - 3′ 0.9 0.01 (120 copies)‡
R455 5′ - CTT AAG GGC CAA AAT TAA AGG ACT G -3′ 0.9
p413S 5′ - 6FAM - TCC TAA CGA ACC TCA - MGBNFQ 0.2
Marburg Ravn-MGB NP EF446131 66 F350 5′ - GGA CGC GGG CTA TGA GTT TG - 3′ 0.9 0.1
R415 5′ - GGA ATA ACC TCT AGA AAG CGA GTT G -3′ 0.9
p371S 6FAM - TGT CAT CAA GAA TCC TG - MGBNFQ 0.2
Marburg Angola-MGB VP40 DQ447660 66 F4573T 5′ - CCA GTT CCA GCA ATT ACA ATA CAT ACA - 3′ 0.6 0.1
R4638G 5′ - GCA CCG TGG TCA GCA TAA GGA - 3′ 0.6
p4601CT 6FAM - CAA TAC CTT AAC CCC C - MGBNFQ 0.2
Marburg Ravn-TM NP DQ447649 77 F1788 5′ - TTA TAT GCT CAG GAA AAG AGA CAG G - 3′ 0.9 0.1
R1864 5′ - CCA ATA CTG CCA AAG GGA TCT TG - 3′ 0.9
p1815S 6FAM - CCC ATA CAG CAT CCA GCC GTG AGC - TAMRA - 3′ 0.1
Marburg Angola-TM NP DQ447660 80 F985 5′ - TCT ATC CTC AGC TCT CAG CAA TTG - 3′ 1.0 0.1
R1064 5′ - TTC GCC GAC ATT GAC ACC AG - 3′ 1.0
p1035A 5′ - 6FAM - TGC CAT GTG CTG TCG CTA CAC CCA - TAMRA - 3′ 0.1
Lassa Josiah-MGB GPC AY628203 80 F3569 5′ - TGC TAG TAC AGA CAG TGC AAT GAG - 3′ 0.9 0.1 (268 copies)‡
R3648 5′ - TAG TGA CAT TCT TCC AGG AAG TGC - 3′ 0.9
p3598S 6FAM - TGT TCA TCA CCT CTT C - MGBNFQ 0.2
Lassa Macenta-MGB NP AY628201 73 F1079 5′ - CAG GAA GGG CAT GGG AAA - 3′ 0.8 10−6 (RNA dilution)§ (257 copies)‡
R1151 5′ - TTG TTG CTC CCA ATT TTT TGT G - 3′ 0.8
p1106S 6FAM - TTG ATT TGG AAT CAG GCG AG - MGBNFQ 0.2
Lassa Weller-MGB NP AY628206 72 F1695 5′ - GCA TTG ATG GAC TGC ATT ATG TTT - 3′ 0.9 0.1 (583 copies)‡
R1766 5′ - CAC AGC TCT TAG GAC CTT TGC AT - 3′ 0.9
p1720S 6FAM - ATG CAG CAG TCT CGG GA - MGBNFQ 0.2
Lassa Pinneo-MGB GPC AY628207 78 F3525 5′ - CCA ATA ATC CCA CAT GTA GCG ATG - 3′ 0.9 0.001
R3602 5′ - GAA CAT TGT GCT AAT TGC GCT TTC - 3′ 0.9
p3559S 6FAM - CCT TCA AGA TTG CCA - MGBNFQ 0.2
Lassa Mozambique (Mopeia)-MGB NP DQ328874 78 F1788 5′ - TCT GGG GAC CGG CAA TTG TG - 3′ 0.9 1.0
R1865 5′ - ACA CCA CAT TGT GCC TTA CTA GAC - 3′ 0.9
p1815S 6FAM - TAT GAC TGC TGC TTC - MGBNFQ 0.2
Lassa Mobala (Acar)-MGB GPC AY342390 80 F1858 5′ - TAC AGA CCA CAG CTA CAC ACA CC - 3′ 1.0 0.001
R1937 5′ - ACT CAC CGT CAC CTG GTT GG - 3′ 1.0
p1915A 6FAM - AGC CGT GCC CAA AG - MGBNFQ 0.2
Lassa Josiah-TM NP AY628203 70 F548 5′ - GGA ATG AGT GGT GGT AAT CAA GG - 3′ 0.6 0.1 (11,711 copies)‡
R617 5′ - TTT TCA CAT CCC AAA CTC TCA CC - 3′ 0.6
p594A 6FAM - ACT CCA TCT CTC CCA GCC CGA GC-TAMRA - 3′ 0.1
Lassa Macenta-TM NP AY628201 73 F1079 5′ - CAG GAA GGG CAT GGG AAA - 3′ 0.8 10−5 (RNA dilution)§ (257 copies)‡
R1151 5′ - TTG TTG CTC CCA ATT TTT TGT G - 3′ 0.8
p1098S 6FAM – CAC TGT TGT TGA TTT GGA ATC AGG CGA GAA G - TAMRA - 3′ 0.1
Lassa Weller-TM NP AY628206 72 F1695 5′ - GCA TTG ATG GAC TGC ATT ATG TTT - 3′ 0.9 0.01 (583 copies)‡
R1766 5′ - CAC AGC TCT TAG GAC CTT TGC AT - 3′ 0.9
p1720S 6FAM - ATG CAG CAG TCT CGG GAG GGC TC-TAMRA - 3′ 0.1
Lassa Pinneo-TM GP AY628207 81 F2730 5′ - CCC AGT TTC CCT TTC CTG AGT - 3′ 0.8 0.1 (234 copies)‡
R2810 5′ - CCA ACG GAG TGT TGC AAA CA - 3′ 0.8
p2757S 6FAM - CAA TGT ATC TTC CAC CCC AGG CCA TTC - TAMRA - 3′ 0.1
Lassa Mozambique (Mopeia)-TM NP DQ328874 81 F2687 5′ - CCT GAT GGT CTC CAG CAT ATT TC - 3′ 0.9 0.1
R2768 5′ - GCT ACA ATT TCA GCT TGT CTG C - 3′ 0.9
p2712S 6FAM - CCC GTC TAT GAG GCA AGC CCC AGC - TAMRA - 3′ 0.1
Lassa Mobala (Acar)-TM GP AY342390 85 F1860 5′ - CAG ACC ACA GCT ACA CAC ACC - 3′ 1.0 0.0001
R1944 5′ - AAT TCC AAC TCA CCG TCA CCT G - 3′ 1.0
p1915A 6FAM - AGC CGT GCC CAA AGC CTC ATC GTC TC - TAMARA - 3′ 0.1
Machupo Carvallo-MGB GPC AY619643 80 F699 5′ - ATG ACC CGT GTG AGG AAG GG - 3′ 0.7 0.001
R778 5′ - GCC ACA GTA GTC AAA GGA ACT GG - 3′ 0.7
p721S 6FAM - AGT GTG CTA CCT GAC CAT - MGBNFQ - 3′ 0.2
Machupo Mallele-MGB GPC AY619645 80 F406 5′ - CAC CTT CCT GAT TCG GGT CTC - 3′ 0.8 0.001
R485 5′ - CAA GGT CTT CTG GTT CAT AGA CTG - 3′ 0.8
p428S 6FAM - AGT GTA TCT GTC CTC A - MGBNFQ - 3′ 0.2
Machupo Carvallo-TM GPC AY619643 78 F681 5′ - ATC TCT TCA GGG GCT TCC ATG - 3′ 0.7 0.001
R758 5′ - TGG GGT CAC CAC ACT GAT TG - 3′ 0.7
p728A 6FAM - AGC ACA CTT TCC CTT CCT CAC ACG G - TAMRA - 3′ 0.1
Machupo Mallele-TM Polymerase AY619642 78 F1009 5′ - TTC GAT CAA CAT TGC AGC TAA ATC - 3′ 0.5 0.001
R1086 5′ - GAT GGT GTA TCG GTT GTT GCA G- 3′ 0.5
p1059A 6FAM - CCA TTG TTC ACC GGG CAG GCC AGT - TAMRA - 3′ 0.1
Junin-MGB NP AY619641 65 F1933 5′ - CAT GGA GGT CAA ACA GCT TCC T - 3′ 0.8 0.001 (234 copies)‡
R1997 5′ - GCC TCC AGA CAT GGT TGT GA - 3′ 0.8
p1956S 6FAM - ATG TCA TCG GAT CCT T - MGBNFQ 0.2
Junin-MGB Polymerase AY619640 68 F2427 5′ - TTG CCA AAT TGA CCC ATC TGT A - 3′ 0.8 0.001 (234 copies)‡
R2494 5′ - GCA ATA TGC CGG GTG TAG TGA - 3′ 0.8
p2452S 6FAM - TAG AGT TCG TCT GAT TTC A - MGBNFQ 0.2
Junin-TM NP AY619641 79 F3032 5′ - CAG TTC ATC CCT CCC CAG ATC - 3′ 1.0 0.0001
R3110 5′ - GGT TGA CAG ACT TAT GTC CAT GAA GA - 3′ 1.0
p3083A 6FAM - TGT TCA ACG AAA CAC AGT TTT CAA GGT GGG - TAMRA - 3′ 0.1
Junin-TM Zinc Binding Protein (Z) AY619640 66 F282 5′ - AGG AAT TCG GAC CTC TGC AA - 3′ 1.0 0.001
R347 5′ - CTC CAC CGG CAC TGT GAT T - 3′ 1.0
p303Sa 6FAM - ATC TGT TGG AAG CCC CTA CCT ACC A - TAMRA -3′ 0.1
Sabia-MGB GP U41071 65 F903 5′ - CGT CAG ATC TTA AGT GCT TTG GAA - 3′ 0.8 0.001
R967 5′ - TTC CGA ATC GTG GTC AAG GT - 3′ 0.8
p928S 6FAM - CAC AGC ACT AGC AAA A - MGBNFQ 0.2
Sabia-MGB Polymerase AY358026 73 F948 5′ - TGT GAT CAT TGG TCG CAG CTA - 3′ 0.7 0.01
R1020 5′ - CTG AGA GGG AAG AAG GCA GTG A - 3′ 0.7
p970S 6FAM - ATC GTT ATC CAT TAG AAT CT - MGBNFQ 0.2
Sabia-TM GP U41071 83 F903 5′ - CGT CAG ATC TTA AGT GCT TTG GAA - 3′ 0.8 0.001
R985 5′ - TTT CAA CAT GTC ACA GAA TTC CG - 3′ 0.8
p959A 6FAM - CGT GGT CAA GGT TAC ATT TTG CTA GTG CTG TGT - TAMRA - 3′ 0.1
Sabia-TM Hairpin Intergenic Region and RNA Polymerase AY358026 79 F433 5′ - CCG GGG TGG TGT GGT TTA - 3′ 0.7 0.01
R511A 5′ - AAG GCT CAA GGG TGT TAC CTG - 3′ 0.7
p460S 6FAM - TTC AAC GGA CTG CTG CTG TCT AAA TAG TCT - TAMRA - 3′ 0.1
Guanarito-MGB GP1 AY129247 73 F485 5′ - TGG ATT CTT GGG TGG ACA ATT AA - 3′ 0.6 0.01
R557 5′ - TAG GCT CAC AGC AGA TTC TTG GA - 3′ 0.6
p513S 6FAM - TGG GAC ATG ACT TTT T - MGBNFQ - 3′ 0.2
Guanarito-MGB Polymerase AY358024 80 F521 5′ - GCT GCC GGA GCT GTC TGA - 3′ 0.7 0.1
R599 5′ - ATG GTG CGA GTT TGT GGA CTT - 3′ 0.7
p542S 6FAM - CAC CAA GTC CCT TAA AG - MGBNFQ - 3′ 0.2
Guanarito-TM GP1 AY129247 73 F485 5′ - TGG ATT CTT GGG TGG ACA ATT AA - 3′ 0.8 0.001
R557 5′ - TAG GCT CAC AGC AGA TTC TTG GA - 3′ 0.8
p532A1 6FAM - CCT CAA AAA GTC ATG TCC CAA TCC C - TAMRA - 3′ 0.1
Guanarito-TM Polymerase AY358024 80 F521 5′ - GCT GCC GGA GCT GTC TGA - 3′ 0.7 0.01
R599 5′ - ATG GTG CGA GTT TGT GGA CTT - 3′ 0.7
p542S 6FAM - CAC CAA GTC CCT TAA AGA TTT GCT ATA GCA GAC AC - TAMRA -3′ 0.1
Hantavirus-MGB (Andes) NP AF291702 67 F41 5′ - GAA TGA GCA CCC TCC AAG AAT TG - 3′ 0.9 10−5 (RNA dilution)§
R107 5′ - CGA GCA GTC ACG AGC TGT TG - 3′ 0.9
p71S 6FAM - ACA TCA CAG CAC ACG A - MGBNFQ 0.2
Hantavirus-MGB (Sin Nombre and NY) NP L37904 71 F26 5′ - CTA CGA CTA AAG CTG GAA TGA GC - 3′ 0.8 10−6 (SN)10−2 (NY)(RNA dilution)§
R96 5′ - GAG TTG TTG TTC GTG GAG AGT G - 3′ 0.8
p59S 6FAM - AAG TGC AAG ACA ACA - MGBNFQ 0.2
*

PCR = polymerase chain reaction; PFU = plaque-forming units; MGB = minor groove binder; NP = nucleoprotein; GP = glycoprotein; VP = virus protein; GPC = glycoprotein precursor.

†

Limit of Detection (LOD) for all assays were measured in PFU/PCR unless otherwise stated; all sensitivities were done in triplicate and were positive in three of three replicates.

‡

Synthetic RNA was used to determine LODs in genome copy number (see Materials and Methods).

§

LODs measured as dilution of stock RNA because PFU information was not available.

Machupo Mallele-TM assay run at 62oC.