Table 1 Primer sequences and protocols for PCR amplification of complete coding SH gene with flanking non-coding sequence (316 nucleotides) and complete coding HN protein sequences. Nucleotide positions are relative to AF338106 (major component Jeryl-Lynn of live vaccine). The M13 primer binding sites used for sequencing are marked in bold
From: Mumps: MMR vaccination and genetic diversity of mumps virus, 2007–2011 in Catalonia, Spain
Primer Name | Primer Sequence (5′ - 3′) | Position (AF338106) |
|---|---|---|
Amplification PCR Protocol | ||
Outer PCR reactions | ||
MuV_0Fa | CAAAACAAATCATATCAAYACAATATCAAG | 6105–6134 |
MuV_0Fb | GGCTTAYATTGCRACYAAAGA | 6066–6086 |
MuV_0Rab | TARGAGTATCTCATTTAGGCC | 8464–8444 |
Thermal profile: 45 °C × 30 min - 95 °C × 15 min - 40 cycles (94 °C × 30 s - 50 °C × 1 min - 68 °C × 3 min) - 68 °C × 10 min | ||
Inner PCR reactions | ||
MuV_SH_1F | TGTAAAACGACGGCCAGTTCRAGTAGTGTCGATGATCTCAT | 6130–6152 |
MuV_SH_1R | CAGGAAACAGCTATGACCTTRCTCAAGCCTTGRTCATT | 6810–6791 |
MuV_HN_2F | TGTAAAACGACGGCCAGTTYCGRACCTGYTTCCGAATA | 6699–6718 |
MuV_HN_2R | CAGGAAACAGCTATGACCACTGTTGCAATYGAGCAG | 7359–7342 |
MuV_HN_3F | TGTAAAACGACGGCCAGTCATAATGTRATTAATGCCAACTG | 7196–7218 |
MuV_HN_3R | CAGGAAACAGCTATGACCCACCAGCTRGTACTYCTCTG | 7893–7874 |
MuV_HN_4F | TGTAAAACGACGGCCAGTTGYATTYCTDGTCTGTGCYTG | 7744–7764 |
MuV_HN_4R | CAGGAAACAGCTATGACCGCCATTCTGGCCTGTT | 8442–8427 |
Thermal profile: 95 °C × 5 min - 35 cycles (95 °C × 30 s - 50 °C × 30 s - 72 °C × 1 min) - 72 °C × 10 min | ||
Sequencing protocol | ||
M13F | TGTAAAACGACGGCCAGT | |
M13R | CAGGAAACAGCTATGACC | |
Thermal profile: 96 °C × 1 min - 30 cycles (96 °C × 10 s - 50 °C × 5 s - 60 °C × 4 min) | ||