Figure 1Amplification patterns of MLVA on Brucella abortus biovar 1. Lane M, 100-bp ladder; Lane P, B. abortus 2308; Lanes 1–20, B. abortus biovar 1 isolates. The indicated PCR products size of the reference strain was confirmed by DNA sequencing. The six VNTR loci (Bruce 01, Bruce 14, Bruce 22, Bruce 30, Bruce 71, and Bruce 72) show differentiation in B. abortus human isolates. MLVA = multilocus variable-number tandem-repeat (VNTR) analysis; PCR = polymerase chain reaction.
Figure 2The stability of 15 VNTR loci on Brucella. Lane M, 100-bp ladder; Lane P, Brucella abortus 2308, isolate 1, isolate 3 positive control; Lanes 1–30, passages. The five VNTR loci (Bruce 09, Bruce 11, Bruce 16, Bruce 42, and Bruce 43) show stability during serial passages. VNTR = variable-number tandem-repeat.
Table 1Primers used for multiplex PCR
Primer name |
5′→3′ sequence∗
|
Bruce 01 |
HEXGGTCTGGGAAAACATGAAAAGC |
Bruce 04 |
6-FAMCTGACGAAGGGAAGGCAATAAG |
Bruce 25 |
NEDGGGAGTATGTTTTGGTTGCACA |
Bruce 11 |
6-FAMCTGTTGATCTGACCTTGCAACC |
Bruce 30 |
NEDTGACCGCAAAACCATATCCTTC |
Bruce 07 |
HEXGCTGACGGGGAAGAACATCTAT |
Bruce 42 |
6-FAMCATCGCCTCAACTATACCGTCA |
Bruce 72 |
NEDGAAGACGGCTATCGACTGGTCT |
Bruce 09 |
HEXGCGGATTCGTTCTTCAGTTATC |
Bruce 16 |
6-FAMACGGGAGTTTTTGTTGCTCAAT |
Bruce 71 |
NEDGAAGACGGCTATCGACTGGTCT |
Bruce 14 |
HEXTTGCTTTATCTTATCTGATTCTTTCAA |
Bruce 22 |
6-FAMGATGAAGACGGCTATCGACTG |
Bruce 43 |
NEDTCTCAAGCCCGATATGGAGAAT |
Table 2Comparison between gel electrophoresis and genotyping assay of Simpson diversity index of each of the 15 VNTR loci with human Brucella abortus isolates
Capillary electrophoresis analysis
|
Locus |
Diversity |
Confidence interval |
K
|
Bruce 22 |
0.871 |
0.847–0.895 |
12 |
Bruce 01 |
0.864 |
0.840–0.888 |
12 |
Bruce 14 |
0.840 |
0.812–0.869 |
11 |
Bruce 72 |
0.829 |
0.793–0.866 |
11 |
Bruce 71 |
0.796 |
0.743–0.848 |
9 |
Bruce 43 |
0.590 |
0.513–0.643 |
7 |
Bruce 25 |
0.578 |
0.513–0.531 |
4 |
Bruce 30 |
0.408 |
0.285–0.531 |
4 |
Bruce 04 |
0.285 |
0.161–0.409 |
4 |
Bruce 07 |
0.140 |
0.039–0.241 |
4 |
Bruce 09 |
0.117 |
0.023–0.211 |
3 |
Bruce 42 |
0.116 |
0.024–0.208 |
2 |
Bruce 11 |
0.095 |
0.008–0.182 |
3 |
Bruce 16 |
0.094 |
0.009–0.179 |
2 |