Volume 17, Number 8—August 2011
Aichi Virus Shedding in High Concentrations in Patients with Acute Diarrhea
|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§|
||Nested, 1st round‡
|AiV-F274||CCAGCCTGACGTATCACAGG||274–293||5′ UTR||+||Real-time¶||Viral RNA quantification
|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).