TABLE 1.
PCR primers used in this study
| Primer | Sequence (5′-3′) | Position (nt)a | PCR product length (bp) |
|---|---|---|---|
| ViPi04 amplificationb | |||
| PCR 1 | |||
| CLONS | AGTTTTCCACGCCCGTCCGC | 114-133 | |
| P1AS | ACGCACTCCTCGCTTACGCTCG | 3075-3095 | |
| UNIVSc | TCAAGGGGCAATTCGGGCT | 205-223 | |
| P3ASc | CTTACGCTCGGAGTGCTTAGTG | 3063-3083 | 2,879 |
| PCR 2 | |||
| MONS1 | TATCACCTACACGCCCCCATTATCCA | 2781-2806 | |
| MONAS2 | TCGCCTCCATCTCCATCGCCAC | 647-668 | |
| MONS3c | GACTGGAAAGAGGAACAGGATG | 2870-2891 | |
| MONAS4c | TTGGGAGCGGGCAGAGCGGG | 550-569 | 1,474 |
| ViPi04 sequencing | |||
| MONSEQ1 | CTGGAGATGGTGGTGGAGACGC | 617-638 | |
| MONSEQ2 | GCTTTCTTCTTCTGAGGTTTGCC | 2626-2648 | |
| MONSEQ3 | AAAGACACTCAAATAACAAG | 1149-1168 | |
| MONSEQ4 | AAATCTAAAGACTATGGGT | 2174-2192 | |
| MONS5 | CGTCACGGCAGCCATTTTA | 3407-3425 | |
| MONAS6 | TACAGCCCAGGAAATGAATC | 54-73 | |
| Group 2 specificd | |||
| G2new1 | TAGACGCCCCAGAGTAAGGAGA | 719-740 | |
| G2new2 | AGTTTTGTTGGTGAAATATGC | 2206-2226 | |
| G2tth2 | GTGAAATAGGCAGATGTACCA | 2196-2216 | |
| G2new3 | CCTGCGGTTTCCGTTCTG | 1361-1378 | 866e |
a
Nucleotide (nt) positions are according to isolate ViPi04.
b
Overlapping PCR fragments encompassing the entire sequence of isolate ViPi04 were obtained by two distinct PCR reactions.
c
Primers also used for sequencing isolate ViPi04.
d
Genogroup 2-specific PCR was performed by using G2new1, G2new2, and G2tth2 in the first step and G2new3, G2new2, and G2tth2 in the second step, respectively.
e
Size of PCR products ranging from 856 to 866 bp depending on the binding of G2tth2 or G2new2 to the target sequence, respectively.