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Volume 14, Number 9—September 2008

Research

Circulation of 3 Lineages of a Novel Saffold Cardiovirus in Humans

Jan Felix Drexler, Luciano Kleber de Souza Luna, Andreas Stöcker, Patrícia Silva Almeida, Tereza Cristina Medrado Ribeiro, Nadine Petersen, Petra Herzog, Célia Pedroso, Hans Iko Huppertz, Hugo da Costa Ribeiro, Sigrid Baumgarte, and Christian DrostenComments to Author 
Author affiliations: Federal University of Bahia, Salvador, Brazil (J.F. Drexler, A. Stöcker, P. Silva Almeida, T.C. Medrado Ribeiro, C. Pedroso, H. da Costa Ribeiro Jr.); Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (J.F. Drexler, L.K. de Souza Luna, N. Petersen, P. Herzog); Professor Hess Paediatric Hospital, Bremen, Germany (H.I. Huppertz); Institute of Hygiene and the Environment, Hamburg (S. Baumgarte); University of Bonn Medical Centre, Bonn, Germany (C. Drosten);

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Table 1

PCR oligonucleotides and formulations for cardiovirus screening*

ID no. Sequence (5′ → 3′) Position† Orientation Usage
CF188 CTAATCAGAGGAAAGTCAGCAT 188–209 + Nested RT-PCR, 1st round‡
CF204 CAGCATTTTCCGGCCCAGGCTAA 204–226 + Nested RT-PCR, 2nd round§
CR718 GCTATTGTGAGGTCGCTACAGCTGT 718–742 Nested RT-PCR, 2nd round§
CR990 GACCACTTGGTTTGGAGAAGCT 990–1011 Nested RT-PCR, 1st round‡
CF723 TGTAGCGACCTCACAGTAGCA 723–743 + Real-time PCR¶
CR888 CAGGACATTCTTGGCTTCTCTA 888–909 Real-time PCR¶
CP797 FAM-AGATCCACTGCTGTGAGCGGTGCAA-BHQ1 797–821 + (probe) Real-time PCR¶

*ID, identification; RT-PCR, reverse transcription–PCR; FAM, 6-carboxyfluorescein; BHQ1, black hole quencher 1.
†Relative to Saffold virus EF165067 genome.
‡25-µL reactions used the QIAGEN OneStep RT-PCR kit (QIAGEN, Hilden, Germany), with 400 nmol/L each of 1st-round primers CF188 and CR990, 1 µL enzyme mix, 1 µg bovine serum albumin, and 5 µL RNA extract. Amplification involved 30 min at 50°C; 15 min at 95°C; 10 cycles of 20 s at 94°C, 30 s starting at 60°C with a decrease of 1°C per cycle, and 50 s at 72°C; and 40 cycles of 20 s at 95°C, 30 s at 54°C, and 50 s at 72°C with a final elongation step of 5 min at 72°C.
§50-µL reactions used 1 µL of 1st-round PCR product, with 1x Platinum Taq buffer (Invitrogen, Karlsruhe, Germany), 200 µmol/L deoxynucleoside triphosphates each, 2.5 mmol/L MgCl2, 400 nmol/L each of 2nd-round primers CF204 and CR718, and 1 U Platinum Taq polymerase. Amplification involved 3 min at 94°C and 45 cycles of 20 s at 94°C, 30 s at 60°C, and 40 s at 72°C.
¶25-µL reactions used 3 µL of RNA extract, 1x reaction buffer and enzymes from the QIAGEN OneStep RT-PCR kit, 600 nM of primer CF723, 400 nM of primer CR888, and 160 nM of probe CP797. Cycling in an Applied Biosystems 7700 SDS instrument involved the following steps: 55°C for 15 min, 95°C for 15 min, and 45 cycles of 95°C for 15 s/58°C for 30 s.

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