[Analytical procedure of variable number of tandem repeats (VNTR) analysis and effective use of analysis results for tuberculosis control].

Variable number of tandem repeats (VNTR) analysis is one of the methods for molecular epidemiological studies of Mycobacterium tuberculosis. VNTR analysis is a method based on PCR, provides rapid highly reproducible results and higher strain discrimination power than the restriction fragment length polymorphism (RFLP) analysis widely used in molecular epidemiological studies of Mycobacterium tuberculosis. Genetic lineage compositions of Mycobacterium tuberculosis clinical isolates differ among the regions from where they are isolated, and allelic diversity at each locus also differs among the genetic lineages of Mycobacterium tuberculosis. Therefore, the combination of VNTR loci that can provide high discrimination capacity for analysis is not common in every region. The Japan Anti-Tuberculosis Association (JATA) 12 (15) reported a standard combination of VNTR loci for analysis in Japan, and the combination with hypervariable (HV) loci added to JATA12 (15), which has very high discrimination capacity, was also reported. From these reports, it is thought that data sharing between institutions and construction of a nationwide database will progress from now on. Using database construction of VNTR profiles, VNTR analysis has become an effective tool to trace the route of tuberculosis infection, and also helps in decision-making in the treatment course. However, in order to utilize the results of VNTR analysis effectively, it is important that each related organization cooperates closely, and analysis should be appropriately applied in the system in which accurate control and private information protection are ensured.

Chromosome arm location of the genes for the biosynthesis of hordatines in barley.

Hordatines A and B, the strong antifungal compounds in barley (Hordeum vulgare), are biosynthesized from p-coumaroyl- and feruloyl-CoA and agmatine by two successive reactions catalyzed by agmatine coumaroyltransferase (ACT) and peroxidase. ACT catalyzes the formation of agmatine conjugates (p-coumaroylagmatine and feruloylagmatine) from precursor CoAs and agmatine, and peroxidase catalyzes the oxidative coupling of agmatine conjugates to form hordatines. Our previous study demonstrated that the short arm of barley chromosome 2H (2HS) is responsible for the biosynthesis of hordatines. In the present study, however, barley genes encoding the ACT (HvACT) and a peroxidase (HvPrx7) were found to be located on the long arm of 2H (2HL). The amounts of hordatines and precursor agmatine conjugates were analyzed in wheat (Triticum aestivum) and wheat lines carrying a whole 2H chromosome, 2HS or 2HL. The addition of 2H and 2HL elevated the levels of agmatine conjugates in wheat. This could be attributed to the HvACT on 2HL. However, the content of agmatine conjugates increased also in the 2HS addition line, suggesting the presence of another unidentified ACT gene on 2HS. Hordatines were detected in wheat, but their content was by far lower than those in barley. The 2H and 2HS addition lines accumulated substantial amounts of hordatines, while the 2HL addition line accumulated them as little as wheat did in spite of the substantial transcription of the HvPrx7 gene on 2HL and of the increased accumulation of the precursor agmatine conjugates. These facts suggest that the HvPrx7 gene on 2HL is not involved in the hordatine biosynthesis and that unidentified peroxidase gene responsible for the hordatine biosynthesis is located on 2HS in barley.

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis.

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.

[Cluster analysis of restriction fragment length polymorphism patterns of Mycobacterium tuberculosis isolated in Chiba prefecture].

Methods for cluster analysis of IS6110 based restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis isolates were studied for an epidemiological investigation in Chiba prefecture. To normalize patterns, external size markers were adopted instead of typical internal size markers used in the standard method. RFLP patterns were run on 1.4% agarose gels and external markers were applied to outside and middle lanes on each gel for precise comparison. The resulting RFLP patterns of 74 isolates were clustered by similarity. Similarity was calculated with the Dice coefficient using parameter settings at 0.8% tolerance and 0.5% optimization. Patterns of 19 isolates from 8 outbreaks showed high similarity within each outbreak. Cluster analysis, as described here, provides insights into epidemiological tracing of tuberculosis in Chiba prefecture.

Comparative analysis of Mycobacterium tuberculosis Beijing strains isolated in three remote areas of Japan.

A quantitative and qualitative comparison was carried out of Mycobacterium tuberculosis Beijing strains isolated in three remote areas of Japan. A total of 452 strains from Chiba Prefecture, 75 from Yamagata Prefecture, and 315 from Kobe City were analyzed for 24 loci by variable number of tandem repeats typing (24(Beijing)-VNTR). All strains were classified in six Beijing subgroups (B(SUB)), B1 to B5 and T, based on a minimum spanning tree reconstructed using data of a standard set of 15 VNTR loci. No significant difference was found in the distribution of strains in the B(SUB) in the three areas, with one exception due to a B5 outbreak in Yamagata, indicating no significant quantitative difference in the B(SUB) in the three areas (P<0.01, Chi-square test). In addition, when strains in each B(SUB) isolated in the three areas were mixed and standardized index of association (I(A)(s)) and variance (Φ(PT)) values were calculated, no significant qualitative difference in the B(SUB) in the three areas was found. These results suggested that the B(SUB) diverged prior to the introduction of M. tuberculosis Beijing strains into Japan. Differences in the distribution of strains in each B(SUB) between Japan and continental Asian countries suggested there had been genetic drift in the continental Asian countries in which B4 had been dominant.

Population genetic analysis of Mycobacterium tuberculosis Beijing subgroup strains.

Population genetic analysis using variable-number tandem repeat (VNTR) data of 23 loci (15 "optimized MIRU" loci and eight "Beijing option" loci) was done on Mycobacterium tuberculosis Beijing lineage strains isolated in Japan. These strains were divided into Beijing subgroups (B(SUB)) B1-B5 and T2 by minimum spanning tree (MST) analysis. The Φ(PT) values among the B(SUB), a measure of their molecular variance, were significantly different from zero with 999 permutations, indicating the validity of B(SUB) classification using the 23 VNTR loci. Higher number of migrants (Nm) values were observed between B1 and T2, B4 and T2, B3 and T2, and B3 and B4 in a phylogenetic network model reconstructed from previously reported single-nucleotide polymorphism (SNP) data. These B(SUB) combinations, except B3 and B4, shared SNP types; i.e., ST19 was in B1 and T2 and in B4 and T2, and STK was in B3 and T2. These results taken together suggested that shared SNP types were not due to homoplasy, but to strong genetic relatedness between those B(SUB). Haploid genetic diversity and standardized index of association values were different in each B(SUB), indicating that the diversity of each B(SUB) was different. Although the differences in B(SUB) diversity were mostly in accordance with the relative divergence order of the B(SUB) in a phylogenetic network model, the diversity of B4 was biased by a significant increase in the number of strains in this study from patients born after 1964 (Fisher's exact test P<0.01). The different diversity of each B(SUB) indicated increased diversity of Beijing lineage strains, perhaps contributing to the survival and dissemination of these strains.

Concordance of variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses of Mycobacterium tuberculosis strains.

Variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses were compared to determine whether VNTR analysis was effective for population genetic analysis of Mycobacterium tuberculosis strains. A total of 682 strains, 510 Beijing genotype and 172 non-Beijing genotype strains, were studied. The number of repeats was investigated for 24 VNTR loci: the 15 loci of "optimized miru", the 8 loci of "Beijing option", and 1 locus for "JATA12". Six loci (miru31, Mtub4, QUB4156c, QUB3232, VNTR3820, and VNTR4120) showed significantly different median numbers of repeats in strains belonging to different lineages defined by LSP (P<0.01, Mann-Whitney U test). When a minimum-spanning tree (MST) was reconstructed using these 6 loci, most strains clustered in the expected branches in the MST branches. However, topology of the MST was not congruent with the evolutional hypothesis of M. tuberculosis, indicating that MST analysis using VNTR data should not use for phylogeny of the organism. When the standardized index of association (sI(A)) was calculated using data for the 6 VNTR loci, the value of sI(A) was significantly different from zero (Monte Carlo simulation with 10,000 resamplings) in every lineage, indicating the linkage disequilibrium in different lineage strains of M. tuberculosis. These results were consistent with the hypothesis that clonal evolution of lineages of the organism has occurred. Therefore, the 6 loci identified in this study would be effective for M. tuberculosis population genetic analysis due to their significantly different median numbers of repeat and linkage disequilibrium though VNTR data was not effective for phylogeny of the organism.