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Volume 17, Number 8—August 2011

Dispatch

Aichi Virus Shedding in High Concentrations in Patients with Acute Diarrhea

Jan Felix Drexler, Sigrid Baumgarte, Luciano Kleber de Souza Luna, Monika Eschbach-Bludau, Alexander N. Lukashev, and Christian DrostenComments to Author 
Author affiliations: Author affiliations: University of Bonn Medical Centre, Bonn, Germany (J.F. Drexler, M. Eschbach-Bludau, C. Drosten); Institute of Hygiene and the Environment, Hamburg, Germany (S. Baumgarte); Bernhard Nocht Institute for Tropical Medicine, Hamburg (L.K. de Souza Luna); Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow, Russia (A.N. Lukashev)

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Table 1

PCR oligonucleotides used for AiV amplification and quantification, Germany, 2004*

ID no. Sequence, 5′ → 3′ Position† Genome location Orientation RT-PCR type Usage
AiV-F65 CACCGTTACTCCATTCAGCTTCTTC 65–89 5′ UTR + Nested, 1st round‡ Determination of suitable genomic target region for quantitative real-time RT-PCR
AiV-F69 GTTACTCCATTCAGCTTCTTCGGAAC 69–94 5′ UTR + Nested, 2nd round§
AiV-R1039 CAGGATTGGACATCAGAATCATAGAG 1039–1064 Leader Nested, 2nd round§
AiV-R1049
GGATAGAACCAGGATTGGACATCAG
1049–1073
Leader

Nested, 1st round‡
AiV-F274 CCAGCCTGACGTATCACAGG 274–293 5′ UTR + Real-time¶ Viral RNA quantification
AiV-R313 AAGCTGCTCACGTGGCAATTGTG 313–335 5′ UTR Real-time¶
AiV-P294
FAM-CTGTGTGAAGYCC-MGBNFQ
294–306
5′ UTR
+ (probe)
Real-time¶
AiV-F2984 CAGGCATTCATCTCYGCAGGTGAA 2984–3007 VP1 + Nested, 1st round‡ Determination of viral genotype
AiV-F2995 CTCYGCAGGTGAATCCTTCAACGT 2995–3018 VP1 + Nested, 2nd round§
AiV-R3881 GATGGCCCAGTGGACGTAGGT 3881–3901 VP1 Nested, 2nd round§
AiV-R3884 TTGCGGATGGCCCAGTGGACGTA 3884–3906 VP1 Nested, 1st round‡

*AiV, Aichi virus; ID, identification; RT-PCR, reverse transcription PCR; UTR, untranslated region.
†Relative to AiV GenBank accession no. AB040749.
‡25-µL QIAGEN OneStep RT-PCR reactions as described by the manufacturer (QIAGEN, Hilden, Germany) used 400 nmol/L each of 1st-round primers, 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; and a final elongation step of 5 min at 72°C.
§50-µL Platinum Taq reactions as described by the manufacturer (Invitrogen, Karlsruhe, Germany) used 1 µL of 1st-round PCR product, 2.5 mmol/L MgCl2, and 400 nmol/L each of 2nd-round primers. 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 QIAGEN OneStep RT-PCR reactions used 3 µL of RNA extract, 600 nmol/L of each primer, and 320 nmol/L of the probe. Cycling in an Applied Biosystems (Darmstadt, German) 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 and 58°C for 30 s (fluorescence measured).

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