TABLE 2.

Primers and RT-PCR conditions used to amplify long-fragment NoV RdRp-capsid-poly(A)-3′ regions from mussel and patient samples

RNA extract sourcePCR methodPrimer pairaPCR conditionsProduct size (bp)Source or reference(s)
Mussel digestive glandCombined RT-PCRNVp36/N7610R43°C (90 min); 95°C (3 min); 5 cycles at 94°C (30 s), 42°C (30 s), 68°C (4 min) + 5 s/cycle; 40 cycles at 94°C (30 s), 50°C (30 s), 68°C (4 min) + 5 s/cycle; 68°C (10 min)3,12335, this study
Seminested PCRbNVp36/GV7As for combined RT-PCR but without the 43°C (90 min) step2,6219,35
GIFFN/N7610R2,2836, this study
Cycle sequencingcGiven in Table 396°C (1 min); 25 cycles at 95°C (15 s), 52°C (5 s), 60°C (4 min)
Patient stoolCombined RT-PCRNVp36/T25VN-3′As for combined RT-PCR with mussel digestive gland3,16719,35
Semi-Nested PCRNVp36/N7610RAs for seminested PCR with mussel digestive gland3,12335, this study
Cycle sequencingcGiven in Table 3As for cycle sequencing with mussel digestive gland
  • a Targeting the RdRp-capsid-poly(A)-3′ region of the NoV genome.

  • b One primer pair used in each master mix.

  • c Unidirectional reactions with individual primers.