Volume 13, Number 7—July 2007
Virus Detection and Monitoring of Viral Load in Crimean-Congo Hemorrhagic Fever Virus Patients
|Oligonucleotide*||Purpose, concentration in nM||Sequence and label (5′→3′)||Position (U88410)†|
|RWCF||Forward primer, 600||CAAGGGGTACCAAGAAAATGAAGAAGGC||1068–1095|
|RWCR||Reverse primer, 600||GCCACAGGGATTGTTCCAAAGCAGAC||1248–1223|
|SE01||Broad-range probe, 100||FAM-ATCTACATGCACCCTGCTGTGTTGACA-TAMRA||1172–1198|
|SE03||Additional probe, 100||FAM-ATTTACATGCACCCTGCCGTGCTTACA-TAMRA||1172–1198|
|SE0A||Additional probe, 100||FAM-AGCTTCTTCCCCCACTTCATTGGAGT -TAMRA||1131–1106|
*All oligonucleotides were used in an assay with the following protocol: 25-µL reaction volume, 5-µL plasma RNA (QIAamp Viral RNA mini kit; QIAGEN, Valencia, CA, USA), 1× concentration of buffer and enzymes from the OneStep RT-PCR kit (QIAGEN), and 400 µmol dNTP, 800-ng nonacetylated bovine serum albumin (Sigma-Aldrich, Munich, Germany). The cycling parameters followed in a Roche LightCycler 1.2 (Roche, Penzberg, Germany) were as follows: 30 min at 50°C, 15 min at 95°C, 46× 15 s at 94°C, and 30 s at 59°C. Fluorescence acquisition occurred at the 59°C step, wavelength filter F1/F2 mode.
†GenBank accession number.