Table 1.
Primer | Nucleotide sequence (5′ to 3′) | DNA target | Programb | Amplicon size |
---|---|---|---|---|
V1 | AAGTGGTGGTGAAGTAACACG | vga(A) | P1 diagnosis | 1 kbp |
V2 | TCAAGAAAGTTTGTTGGTTCATC | |||
T1 | ATATCCGACATCGCCAACTC | Tn5406 | P1 diagnosis | 740 bp |
T2 | TTCGTGTTACTTCACCACCAC | |||
X | ATTTCATTATCGCCATCTGTC | EES57021 | P2 cloning | 2.4 kbp |
Y | TCTTCCTTCTTCAATTTCCC | |||
lnuACDf | GGTTWGATGGWGGYTGGGG | lnu(A) | P3 diagnosis | 230 bp |
lnuAr | AATTGCCACCTTCTGGGTTTGC | |||
lnuBFf | AGARGGTGACSARTWCTCTGA | lnu(B) | P3 diagnosis | 400 bp |
lnuBFr | AKGMRCGAGCATAYTCTCC |
The primer pairs described here were not found in the literature. Other primer pairs were vgaB1 and vgaB2 for detection of vga(B) (24), vgaC-1 and vgaC-2 for detection of vga(C) (21), vgaE_fw and vgaE_rv for detection of vga(E) (22), lsaB-1 and lsaB-2 for detection of lsa(B) (21), cfr_fw and cfr_rv for detection of cfr (23), vatA1 and vatA2 for detection of vat(A) (24), and 16S-27F and 16S-907R for detection of rrs (12).
Thermocycling programs were as follows: P1, 1 cycle of 5 min at 94°C and 2 min at 54°C; 30 cycles of 60 s at 72°C, 60 s at 94°C, and 60 s at 54°C; and 1 cycle of 10 min at 72°C; P2, 1 cycle of 5 min at 94°C and 2 min at 54°C; 30 cycles of 2 min at 72°C, 1 min at 94°C, and 1 min at 54°C; and 1 cycle of 10 min at 72°C); P3, 1 cycle of 5 min at 94°C and 2 min at 50°C; 30 cycles of 40 s at 72°C, 50 s at 94°C, and 50 s at 50°C; and 1 cycle of 10 min at 72°C.