Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis

Bovine tuberculosis caused by Mycobacterium bovis remains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish M. bovis isolates demonstrated that a significant proportion of isolates ( approximately 20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the M. bovis genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on AluI restriction-digested M. bovis genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish M. bovis isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS6110, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype M. bovis by RFLP analysis is compelling.     

Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats.

Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.     

Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.     

Evaluation of the Mycobacterium bovis Restriction Fragment Length Polymorphism Probe pUCD, in Combination with the Direct Repeat Probe, for Molecular Typing of Mycobacterium tuberculosis Strains in Ireland

A mycobacterial restriction fragment length polymorphism probe, pUCD, has recently been described which represents an effective tool for the strain typing of Mycobacterium bovis. The present study evaluated this probe, in combination with the direct repeat probe (DR), for the molecular typing of 90 strains of Mycobacterium tuberculosis from 87 patients, looking at a group (62 isolates) of nonselected samples to assess pUCD combined with DR as a general tool and a subset of 32 isolates with a common specific IS6110 strain type in Ireland. Within the group of 62 isolates, pUCD-DR identified 42 strains and was comparable to both IS6110 (41 strains) and polymorphic guanine-cytosine-rich sequence (PGRS) (37 strains) analysis. pUCD-DR was found to be comparable to IS6110 and PGRS in identifying four separate clusters of isolates which were confirmed to be clinically related. pUCD-DR divided the common IS6110 isolates into six distinct types and was comparable to PGRS (seven strain types). The usefulness of this probe as an epidemiological tool is discussed.     

Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping

Various genetic markers have been exploited for fingerprinting the Mycobacterium tuberculosis complex (MTBC) in molecular epidemiological studies, mainly through identifying restriction fragment length polymorphisms (RFLP). In large-scale studies, RFLP typing has practical processing and analysis limitations; therefore, attempts have been made to move towards PCR-based typing techniques. Spoligotyping (spacer oligotyping) and, more recently, variable-number tandem repeat (VNTR) typing have provided PCR-derived typing techniques. This study describes the identification and characterization of novel VNTR loci, consisting of tandem repeats in the size range of 53 to 59 bp in the MTBC, and their assessment as typing tools in 47 Mycobacterium bovis field isolates and nine MTBC strains. Spoligotyping and the previously described set of exact tandem repeats (ETRs) (R. Frothingham and W. A. Meeker-O'Connell, Microbiology 144:1189-1196, 1998) were also applied to the same panel of isolates. The allelic diversity of the individual VNTR loci was calculated, and a comparison of the novel VNTRs was made against the results obtained by spoligotyping and the existing set of ETRs. Eleven unique spoligotypes were discriminated in the panel of 47 M. bovis isolates. Greater resolution was obtained through the combination of the most-discriminating VNTRs from both sets. Considerable discrimination was achieved, with the 47 M. bovis isolates resolved into 14 unique profiles, while all nine MTBC isolates were uniquely differentiated. The novel VNTR markers described increased the discrimination possible in strain typing of M. bovis, with the added benefit of an intuitive digital nomenclature, with the allele copy number of the individual VNTRs providing a profile. VNTR typing was shown to be a valuable technique with great potential for further development and application to epidemiological tracing of tuberculosis transmissions.     

Differentiation of Mycobacterium bovis isolates from animals by DNA typing.

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.     

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.

Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.     

Molecular typing by random amplification of polymorphic DNA and M13 southern hybridization of related paired isolates of Aspergillus fumigatus.

Three forms of DNA-based typing procedures for Aspergillus fumigatus isolates have been developed over the last five years. The procedures are random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) detection, and Southern hybridizations with various repetitive sequence-based probes. Using two of these procedures, we compared 16 selected isolates, grouped into eight pairs on the basis of epidemiology or previously assigned RFLP types. RAPD with four primers (R108, RC08, 2, and 4), including three previously used with A. fumigatus, showed that one primer, R108, gave the best discrimination (8 types). Southern hybridization of total genomic DNA digested with HindIII and probed with the total bacteriophage M13 genome resulted in the highest overall level of discrimination. Combination of the RAPD and Southern hybridization with the previously assigned RFLP types discriminated 10 isolates of 16. Isolates closely linked epidemiologically could not be distinguished from each other. In addition, three pairs of isolates previously unlinked by epidemiology had the same overall types. Two pairs were obtained from the same hospital within 2 years of each other, whereas the third pair were isolated from California and Germany. A full understanding of the epidemiology and ecology of A. fumigatus requires multiple discriminatory typing procedures.     

Evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment length polymorphism analysis and spoligotyping

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, [corrected] taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen.     

Molecular typing of Mycobacterium bovis isolates from Cameroon

In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.     

Usefulness of Spoligotyping in Molecular Epidemiology of Mycobacterium bovis-Related Infections in South America

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.     

Evaluation of Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis

DNA fingerprinting techniques were used to type 273 isolates of Mycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.     

DNA restriction fragment length polymorphisms characteristic for Dw subtypes of DR2

DNA restriction fragment length polymorphisms (RFLP) can be easily demonstrated in DNA of cells expressing different DR specificities when class II cDNA probes are used for hybridization. Previous studies of DR4+ homozygous typing cells (HTCs) carrying different Dw subtypes, however, detected no RFLP correlating with subtypes. In contrast, we report here Southern blotting studies of DR2+ HTCs carrying different subtypes which showed RFLP patterns characteristic for each subtype, using both DR beta and DQ beta probes and several restriction enzymes. The RFLP between subtypes of DR2 was, however, appreciably lower than that found between DR specificities.     

Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization.     

Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis.

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries.     

Discriminatory power of typing schemes based on Simpson's index of diversity for Neisseria gonorrhoeae.

Simpson's index of diversity was used to produce a single numerical value to compare the abilities of single or combined typing schemes to discriminate between unrelated isolates. This calculation was used to compare the discriminating power of auxotype and serovar determination and plasmid content analysis, either singly or in combination, for Neisseria gonorrhoeae isolates having different antimicrobial susceptibilities (i.e., antibiotic-susceptible isolates and those that produce penicillinase, carry plasmid-mediated resistance to tetracycline, have chromosomally mediated penicillin resistance, or both produce penicillinase and carry plasmid-mediated resistance to tetracycline). Plasmid content analysis and auxotype determination produced the lowest level of discrimination, while a combination of auxotype and serovar typing schemes generally provided higher levels of discrimination. Addition of plasmid content analysis to auxotype and serovar typing provided additional discrimination only with penicillinase-producing isolates. For isolates that carried plasmid-mediated resistance to tetracycline, isolates that were tetracycline resistant, isolates that both produced penicillinase and carried plasmid-mediated resistance to tetracycline, or isolates that had chromosomally mediated penicillin resistance, none of the typing methods produced high discriminatory indices, indicating that these isolates are probably derived from relatively few clones.     

Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.     

Evaluation of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Analysis and Spoligotyping for Genotyping of Mycobacterium bovis Isolates and a Comparison with Restriction Fragment Length Polymorphism Typing

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.The development of molecular techniques for differentiation of Mycobacterium bovis isolates has been of considerable benefit in epidemiological studies. Typing methods that have been commonly used include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (7, 19).RFLP analysis of M. bovis isolates has commonly utilized polymorphism of the insertion sequence IS6110 and repetitive DNA elements such as the polymorphic GC-rich sequence (PGRS) and the direct-repeat (DR) region. Analysis of polymorphism of IS6110, the PGRS, and the DR region in combination has provided a high level of discrimination between strains (7, 19). REA has been widely used in New Zealand and has also given excellent resolution of strains (4). However, both RFLP analysis and REA require relatively large quantities of DNA and are laborious and time-consuming procedures. Complex banding patterns make analysis and interlaboratory comparisons difficult. Spoligotyping is a PCR-based typing method that reveals the presence or absence of unique spacer sequences located between the direct-repeat sequences of the DR region (12). It is a relatively easy procedure to perform, and the results can be expressed in a digital format. However, spoligotyping does not differentiate M. bovis strains to the same extent as RFLP analysis or REA (7, 19). Minisatellite-like loci in the Mycobacterium tuberculosis complex genome, described as mycobacterial interspersed repetitive units, may show polymorphism of the number of tandem repeats. A wide range of M. tuberculosis complex MIRU-VNTR loci have been evaluated, and loci which are informative for M. bovis isolates have been identified (8, 16, 17, 20, 23). Similar to spoligotyping, MIRU-VNTR typing has the advantages of ease of procedure and the generation of results in a digital format.In recent years, genotyping by IS6110, PGRS, and DR RFLP has been used in epidemiological studies of M. bovis infection in the Republic of Ireland (5, 6, 14). While RFLP analysis has given a high level of strain differentiation, its replacement by MIRU-VNTR typing or by a combination of MIRU-VNTR typing and spoligotyping offers potential advantages. The objective of this study was to evaluate MIRU-VNTR typing or a combination of MIRU-VNTR typing and spoligotyping for discrimination of M. bovis strains, to compare the discriminatory powers of the two methods against RFLP analysis, and to investigate the level of concordance between the three typing systems.     

Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis and may represent a feasible approach to study global M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating drug-resistant M. tuberculosis strains of the Beijing genotype family, 102 multidrug-resistant (MDR) clinical isolates and 253 randomly selected non-MDR isolates collected from 2000 to 2003 in Hong Kong were subjected to 12-locus MIRU typing, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. Spoligotyping showed that 243 (68.5%) of 355 isolates belonged to Beijing family genotype. MIRU typing showed lower discrimination in differentiating between the Beijing family strains (Hunter-Gaston discriminative index [HGI] of 0.8827) compared with the IS6110 RFLP method (HGI = 0.9979). For non-Beijing strains, MIRU typing provided discrimination (HGI = 0.9929) comparable to that of the RFLP method (HGI = 0.9961). There was no remarkable difference in discrimination power between the two methods in differentiating both within and between MDR and non-MDR strains of M. tuberculosis. Dendrograms constructed with the MIRU typing data showed a clear segregation between the Beijing and non-Beijing genotype. Addition of RFLP to MIRU typing offered a higher discrimination ability (92.6%) than did addition of MIRU typing to RFLP (40.0%). This supported the potential use of this method to analyze the global genetic diversity of MDR M. tuberculosis strains that may be at different levels of evolutionary divergence.     

Southern hybridization analysis of DNA polymorphism in the HLA-D region

Restriction fragment-length polymorphisms (RFLP) were systematically analyzed by Southern hybridization with restriction endonuclease-digested genomic DNA from 28 HLA-homozygous B cell lines with Dw1-Dw19 specificity using the DR beta and DQ beta chain cDNAs as probes. These probes detected polymorphic fragments unique to each HLA-DR specificity. Furthermore, the DQ beta chain probes permitted us to distinguish between different Dw specificities with an identical DR type much more efficiently than with the DR beta chain probe. Distribution analysis of restriction fragments hybridizing to DR beta in relation to the DR and DQ specificities showed several sets of them forming ten clusters, some of which correlate with DRw53, DQw1, and DR alleles. This DNA typing technique allows the direct definition of HLA types at the gene level and provides a powerful tool for isolating genes controlling HLA-associated diseases.