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Abstract
Background:
Mycobacterium tuberculosis is one of the most clinically significant infectious agents. Especially during mass outbreaks, accurate identification and monitoring are required. The proportion of Beijing family members is very high among infecting strains, and spoligotyping is not suitable for strain typing. Therefore, we studied the homogeneity of isolates using the mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method and identified its utility for carrying out molecular epidemiologic analysis.
Methods:
Eighty-one clinical M. tuberculosis isolates that had previously been analyzed by spoligotyping were used in this study. We used the 12 standard MIRU loci and further four exact tandem repeat (ETR) loci (ETR-A, -B, -C, and -F). Four strains each of randomly selected Beijing and Beijinglike families were subjected to IS6110- restriction fragment length polymorphism analysis.
Results:
All 81 samples showed amplification products of all VNTR loci, and all of them showed differences in at least one locus. The calculation of the Hunter-Gaston diversity index (HGDI) for MIRU-VNTR gave the value of 0.965. Discriminatory index in the six loci (MIRU-10, −16, −26, −31, −39, and ETR-F) were found to be highly discriminated (HGDI >0.6). Beijing and Beijing-like family isolates were discriminated into different MIRU-VNTR types.
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Fig. 1.
MIRU-VNTR electrophoresis patterns of three M. tuberculosis isolates. Lane M, 100-bp molecular marker. Other lanes in each isolate represent the 16 VNTR loci. Order of the 16 VNTR loci is the same as in Table 1 and 2.
Abbreviation: See Table 1.
Fig. 2.
Comparison of molecular strain typing patterns of four M. tuberculosis Beijing family isolates showing the diversity of MIRU-VNTR. (A) IS6110 RFLP analysis pattern, (B) Spoligotyping pattern, and (C) MIRU-VNTR pattern. Order of the 16 VNTR loci is the same as in Table 1 and 2. All isolates belonging to the Beijing family defined by Kremer et al. [21] hybridized to the last nine probes in spoligotyping, but differed in at least three loci in MIRU-VNTR analysis.
Abbreviations: RFLP, restriction fragment length polymorphism; MIRU-VNTR, mycobacterial interspersed repetitive units-variable number of tandem repeats.
Fig. 3.
Comparison of molecular strain typing patterns of four M. tuberculosis Beijing-like family isolates showing diversity of MIRU-VNTR. (A) IS6110 RFLP analysis pattern, (B) Spoligotyping pattern, and (C) MIRU-VNTR pattern. Order of the 16 VNTR loci is the same as in Table 1 and 2. All isolates belonging to the Beijing-like family defined by Kremer et al. [21] hybridized exclusively with most of the last nine probes in spoligotyping, but differed from each other in at least two loci in MIRU-VNTR analysis.
Abbreviations: See Fig. 2.
Table 1.
Primer sequences for amplifying MIRU-VNTR loci
Table 2.
Distribution of 81 M. tuberculosis isolates containing variable numbers of each VNTR locus and allelic diversity
| VNTR Loci | N of VNTR repeats | HGDI∗ | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | ||
| MIRU-2 | 18 | 61 | 2 | 0.388 | |||||||
| MIRU-4 | 1 | 3 | 77 | 0.096 | |||||||
| MIRU-10 | 4 | 35 | 34 | 8 | 0.633 | ||||||
| MIRU-16 | 2 | 32 | 35 | 12 | 0.643 | ||||||
| MIRU-20 | 4 | 77 | 0.095 | ||||||||
| MIRU-23 | 6 | 69 | 4 | 1 | 1 | 0.269 | |||||
| MIRU-24 | 78 | 3 | 0.072 | ||||||||
| MIRU-26 | 1 | 12 | 12 | 9 | 12 | 28 | 5 | 2 | 0.808 | ||
| MIRU-27 | 1 | 23 | 50 | 7 | 0.537 | ||||||
| MIRU-31 | 1 | 8 | 17 | 24 | 20 | 11 | 0.789 | ||||
| MIRU-39 | 10 | 34 | 30 | 7 | 0.672 | ||||||
| MIRU-40 | 1 | 25 | 47 | 7 | 1 | 0.567 | |||||
| ETR-A | 2 | 5 | 73 | 1 | 0.185 | ||||||
| ETR-B | 3 | 72 | 6 | 0.206 | |||||||
| ETR-C | 1 | 5 | 74 | 1 | 0.163 | ||||||
| ETR-F | 1 | 10 | 33 | 26 | 10 | 1 | 0.709 | ||||
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