TABLE 1.

Primers and RT-PCR conditions used in screening mussel digestive gland and patient stool samples for NoV RNA

RNA extract sourcePCR methodPrimer pairaPCR conditionsProduct size (bp)Reference(s)
Mussel digestive glandCombined RT-PCRNVp36/NVp11043°C (1 h); 95°C (3 min); 5 cycles at 94°C (20 s), 45°C (20 s), 72°C (20 s) + 1 s/cycle; 40 cycles at 94°C (20 s), 52°C (20 s), 72°C (20 s) + 1 s/cycle39817,35
Nested PCRJV12Y/JV13I95°C (3 min); 5 cycles at 94°C (20 s), 37°C (10 s), 72°C (20 s); 40 cycles at 94°C (20 s), 42°C (20 s), 72°C (20 s) + 1 s/cycle32833
Cycle sequencingbJV12Y, JV13I96°C (1 min); 25 cycles at 95°C (15 s), 52°C (5 s), 60°C (4 min)33
Patient stoolCombined RT-PCRcNI/NVp11043°C (1 h); 95°C (3 min); 40 cycles at1208,17
NVp36/NVp110d    95°C (20 s), 54°C (30 s), 72°C (30 s)39817,35
NVp69/NVp11011017,35
Nested PCRdJV12Y/JV13I95°C (3 min); 5 cycles at 94°C (20 s), 50°C (10 s), 72°C (20 s); 40 cycles at 94°C (20 s), 54°C (20 s), 72°C (20 s)32833
Cycle sequencingbJV12Y, JV13IAs for cycle sequencing with mussel33
NVp36, NVp110    digestive gland17,35
  • a Targeting the RdRp gene region.

  • b Unidirectional reactions with individual primers.

  • c One primer pair used in each combined RT-PCR master mix.

  • d Selected for nesting of NI/NVp110 or NVp69/NVp110 gel-positive samples.