Variable Number Tandem Repeat Typing Research Articles

Use of the VNTR typing technique to determine the origin of Mycobacterium tuberculosis strains isolated from Filipino patients in Korea

With increasing international interchange of personnel, international monitoring is necessary to decrease tuberculosis incidence in the world. This study aims to develop a new tool to determine origin of Mycobacterium tuberculosis strains isolated from Filipino patients living in Korea. Thirty-two variable number tandem repeat (VNTR) loci were used for discrimination of 50 Filipino M. tuberculosis strains isolated in the Philippines, 317 Korean strains isolated in Korea, and 8 Filipino strains isolated in Korea. We found that the VNTR loci 0580, 0960, 2531, 2687, 2996, 0802, 2461, 2163a, 4052, 0424, 1955, 2074, 2347, 2401, 3171, 3690, 2372, 3232, and 4156 had different mode among copy numbers or exclusively distinct copy number in VNTR typing between Filipino and Korean M. tuberculosis strains. When these differences of the VNTR loci were applied to 8 Filipino M. tuberculosis strains isolated in Korea, 6 of them revealed Filipino type while 2 of them had Korean type. Using the differences of mode or repeated number of VNTR loci were very useful in distinguishing the Filipino strain from Korean strain.

Optimal Combination of VNTR Typing for Discrimination of IsolatedMycobacterium tuberculosisin Korea

BackgroundVariable-number tandem repeat (VNTR) typing is a promising method to discriminate the Mycobacterium tuberculosis isolates in molecular epidemiology. The purpose of this study is to determine the optimal VNTR combinations for discriminating isolated M. tuberculosis strains in Korea.MethodsA total of 317 clinical isolates collected throughout Korea were genotyped by using the IS6110 restriction fragment length polymorphism (RFLP), and then analysed for the number of VNTR copies from 32 VNTR loci.ResultsThe results of discriminatory power according to diverse combinations were as follows: 25 clusters in 83 strains were yielded from the internationally standardized 15 VNTR loci (Hunter-Gaston discriminatory index [HGDI], 0.9958), 25 clusters in 65 strains by using IS6110 RFLP (HGDI, 0.9977), 14 clusters in 32 strains in 12 hyper-variable VNTR loci (HGDI, 0.9995), 6 clusters in 13 strains in 32 VNTR loci (HDGI, 0.9998), and 7 clusters in 14 strains of both the 12 hyper-variable VNTR and IS6110 RFLP (HDGI, 0.9999).ConclusionThe combination of 12 hyper-variable VNTR typing can be an effective tool for genotyping Korean M. tuberculosis isolates where the Beijing strains are predominant.

Estimation of Recent Transmission of Mycobacterium Tuberculosis Strains among Iranian and Afghan Immigrants: A Cluster-Based Study.

Iran has extended borders with high-TB burden countries (Afghanistan and Pakistan) and immigrations of these populations influences TB distribution in the region and threatens the control strategies. The aim of this study was to evaluate the extent of recent TB transmission among Iranian and Afghan cases. Spoligotyping and 15-locus variable number tandem repeat (VNTR) typing were applied to genotype 102 MTB isolates (2009 to 2010). Phylogenetic relationships were analysed by two methods: a cluster-graph method and a minimum spanning tree (MST) method. Furthermore, evaluation of recent TB transmission was assessed with three indices including, RTIn, RTIn-1 and TMI. Using molecular typing, 35 different spoligotypes were detected among the studied isolates. Seventy seven cases (75.4%) were distributed into 10 clusters and the remaining 25 (24.5%) isolates had a unique pattern. The cluster sizes also ranged from 2 to 21 isolates. The most frequent spoligotype in our populations belong to Haarlem (n=30, 29.4%) followed by CAS (n= 29, 28.4%) and Beijing (n=16, 15.6%) lineages. The used indices give the following values: RTIn = 0.75, RTIn-1 = 0.65 and TMI = 0.24. The low rate of TB transmission in our findings (24%) showed that the mode of TB transmission in Iran is mostly associated with reactivation of a previous TB infection and that recently a transmitted disease has a minor role. However, the increasing incidence of the intra-community transmission in recent years highlights the need for establishing new strategies for control of TB.

Genotypic characteristics of Mycobacterium tuberculosis isolated from household contacts of tuberculosis patients in the Philippines

BackgroundThe Philippines has an extremely high rate of tuberculosis but little is known about M. tuberculosis genotypes and transmission dynamics in this country. The aim of this study was to determine the proportion of household contacts who develop active TB due to direct transmission from an index case in that household.MethodsMycobacterium tuberculosis isolates from household contacts of tuberculosis patients in the Philippines were characterized using restriction-fragment-length polymorphism analysis, spoligotyping, and mycobacterial interspersed repetitive units – variable number tandem repeats typing (12-loci) to determine their utility in elucidating transmission in an area of high tuberculosis prevalence. Drug susceptibility patterns for these isolates were also determined.ResultsSpoligotyping and MIRU-VNTR typing results matched in 10 (62.5%) of 16 index patient-household contact pairs while IS6110 fingerprints matched in only six (37.5%) pairs. Only 3/16 (18.8%) index patient-household contact pairs had identical drug susceptibility results.ConclusionsStrain typing of M. tuberculosis isolates from household contacts in the Philippines indicates that transmission of strains does not necessarily occur directly from the index patient living in close proximity in the same household but rather that community-based transmission also frequently occurs. Accurate susceptibility testing of all isolates is necessary to insure optimal care of both the index patients and any culture-positive household contacts.

Differences in the population of genetics of Mycobacterium tuberculosis between urban migrants and local residents in Beijing, China

Background Currently, migration has become one of the risk factors of high burden of tuberculosis in China. This study was to explore the influence of mass migration on the dynamics of Mycobacterium (M.) tuberculosis in Beijing, the capital and an urban area of China. Methods Three hundred and thirty-six M. tuberculosis strains from the Changping district, where the problem of urban migrants was more pronounced than in other Beijing regions, were genotyped by Spoligotyping, large sequence polymorphisms (LSPs 105 and 181), and variable number tandem repeat (VNTR) typing. Based on the genotype data, the phylogeny of the isolates was studied. Results In Changping district, the proportion of Beijing lineage M. tuberculosis isolates amounted to 89.0% (299/336), among which 86.6 % (252) belonged to the modern lineage. The frequency of modern Beijing lineage strains is so high (around 75% (252/336)) that associated risk factors affecting the tuberculosis epidemic cannot be determined. The time to the most recent common ancestor (TMRCA) of the Beijing lineage strains was estimated to be 5073 (95% CI: 4000-6200) years. There was no significant difference in the genetic variation of Beijing isolates from urban migrants and local residents. Conclusions The clone of modern Beijing lineage M. tuberculosis, which is dominant in the Beijing area, most likely started to expand with the five thousand-year-old Chinese civilization. In the future, with the urbanization in the whole of China, modern Beijing lineage M. tuberculosis may gain the larger geographical spread.

Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam

The molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular tests. We quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics. Consecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP. Among 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found. Strains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.

Evaluation of the Discriminatory Power of Variable Number of Tandem Repeat Typing ofMycobacterium bovisIsolates from Southern Africa

The usefulness of variable number tandem repeat (VNTR) typing based on limited numbers of loci has previously proven inferior compared to IS6110-RFLP typing when applied to the study of the molecular epidemiology of bovine tuberculosis (BTB) in both livestock and wildlife in southern Africa. In this study, the discriminatory power of 29 published VNTR loci in the characterization of 131 Mycobacterium bovis strains isolated predominantly from wildlife and a smaller number from livestock in southern Africa was assessed. Allelic diversities calculated when loci were evaluated on a selected panel of 23 M. bovis isolates with identified varying degrees of genetic relatedness from different geographic origins as well as M. bovis BCG ranged from 0.00 to 0.63. Of the 29 loci tested, 13 were polymorphic (QUB 11a, QUB 11b, QUB 18, ETR-B and -C, Mtub 21, MIRU 16 and 26, ETR-E, QUB 26, MIRU 23, ETR-A, and Mtub 12). In addition, a comparative evaluation of the 13 loci on a panel of 65 isolates previously characterized by IS6110 restriction fragment length polymorphism (RFLP) typing and further evaluation on 41 isolates with no typing history from Kruger National Park (KNP) highlighted that M. bovis from epidemiologically unrelated cases of BTB in different geographic regions can be adequately distinguished. However, there is a need for improvement of the method to fully discriminate between the parental KNP strain and its clones to allow the detection of evolutionary events causing transmission between and within wildlife species.

Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

Genotypic characterization by spoligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia

This work is an approach to the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were genotyped by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the country, and SB2025 (Mycobacterium bovis) from Sfax Region (Southern Tunisia). A second M. caprae isolate with a spoligotyping profile previously described in Europe mainland, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribution of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions selected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M. bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diversity of the MTBC in Tunisia.

Qualitative Analysis To Ascertain Genotypic Identity of or Differences between Mycobacterium tuberculosis Isolates in Laboratories with Limited Resources

Mycobacterium tuberculosis is currently genotyped using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, although the high cost of this technique restricts its implementation in resource-limited settings. We designed a MIRU-VNTR format, MLP3 (MIRU-VNTR length polymorphism triplex), that is based on the qualitative comparison of 5 nonfluorescent 3-band fingerprints in conventional electrophoresis and minimizes costs and technical demands. MLP3 successfully resolved cross-contamination alerts, discriminated reinfections from reactivations, clarified suspected microepidemics, and tracked transmission events of high epidemiological interest.

Mycobacterium tuberculosisis the causative agent of tuberculosis in the southern ecological zones of Cameroon, as shown by genetic analysis

BackgroundTuberculosis (TB) is a major cause of mortality and suffering worldwide, with over 95% of TB deaths occurring in low- and middle-income countries. In recent years, molecular typing methods have been widely used in epidemiological studies to aid the control of TB, but this usage has not been the case with many African countries, including Cameroon. The aims of the present investigation were to identify and evaluate the diversity of the Mycobacterium tuberculosis complex (MTBC) isolates circulating in two ecological zones of Cameroon, seven years after the last studies in the West Region, and after the re-organization of the National TB Control Program (NTBCP). These were expected to shed light also on the transmission of TB in the country. The study was conducted from February to July 2009. During this period, 169 patients with symptomatic disease and with sputum cultures that were positive for MTBC were randomly selected for the study from amongst 964 suspected patients in the savannah mosaic zone (West and North West regions) and the tropical rainforest zone (Central region). After culture and diagnosis, DNA was extracted from each of the MTBC isolates and transported to the BecA-ILRI Hub in Nairobi, Kenya for molecular analysis.MethodsGenetic characterization was done by mycobacterial interspersed repetitive unit–variable number tandem repeat typing (MIRU-VNTR) and Spoligotyping.ResultsMolecular analysis showed that all TB cases reported in this study were caused by infections with Mycobacterium tuberculosis (98.8%) and Mycobacterium africanum (M. africanum) (1.2%) respectively. We did not detect any M. bovis. Comparative analyses using spoligotyping revealed that the majority of isolates belong to major clades of M. tuberculosis: Haarlem (7.6%), Latin American-Mediterranean (34.4%) and T clade (26.7%); the remaining isolates (31.3%) where distributed among the minor clades. The predominant group of isolates (34.4%) corresponded to spoligotype 61, previously described as the “Cameroon family. Further analysis based on MIRU-VNTR profiles had greater resolving power than spoligotyping and defined additional genotypes in the same spoligotype cluster.ConclusionThe molecular characterization of MTBC strains from humans in two ecological regions of Cameroon has shown that M. tuberculosis sensu stricto is the predominant agent of TB cases in the zones. Three decades ago, TB was reported to be caused by M. africanum in 56.0% of cases. The present findings are consistent with a major shift in the prevalence of M. tuberculosis in Cameroon.

Genotypic characteristics of Mycobacterium avium subsp. hominissuis strains

Mycobacterium avium are typical environmental, non-tuberculosis microorganisms that occasionally cause mycotuberculosis, an infectious disease in wild and domestic animals, birds, and humans. Here, we report the results of the first study on the genetic diversity of the Russian population of M. avium. A total of 85 M. avium subsp. hominissuis (MAH) clinical strains were isolated from patients (including 30 HIV-positive individuals) with mycobacteriosis in St. Petersburg, 2008-2011. The biochemical identification of the microorganisms was carried out using the PCR detection of the mobile elements IS901 and IS900, as well as of the polymorphism of restriction fragments of the hsp65 gene. The genetic diversity of the isolates was evaluated by VNTR typing based on eight variable-number tandem repeats (VNTRs) (292, X3, 25, 47, 3, 7, 10, and 32 [Thibault et al., 2007]). The MAH population studied was characterized by 15 VNTR types, including nine unique patterns and six clusters of isolates with identical eight-digit profiles. The largest clusters (22221128 and 24221128) included 45 (59.2%) and 15 (19.7%) isolates, respectively; the others contained from 2-7 strains. The strains of the cluster 2533112'8 possessed a truncated TR10 locus (allele 2'). Taking into account the absence of the epidemiological links between the patients and the fact that the infection was delivered from the environment, the high rate of clustering of MAH isolates can be explained by the low discriminatory power of the eight-locus VNTR-typing scheme (HGDI 0-0.61).

Multiplex variable number of tandem repeats for Oenococcus oeni and applications

Oenococcus oeni is responsible for the malolactic fermentation of wine. Genomic diversity has already been established in this species. In addition, winemakers usually report varying starter-culture efficiency. It is essential to monitor indigenous and selected strains in order to understand strain survival and development during the winemaking process. A previous article described a variable number of tandem repeats (VNTR) scheme, based on five polymorphic loci of the genome. VNTR typing of O. oeni was highly discriminating, faster, and more reliable than the PFGE or MLST methods. The objective of this study was to set up a faster protocol by multiplexing, taking advantage of the high performance of multicolor capillary electrophoresis. The primers were labeled with multiple fluorescent dyes. PCR conditions were adapted by multiplexing amplifications in two separate PCR mixtures for the five loci, both at the same annealing temperature. The resulting assay proved to be robust, accurate, fast and easy to perform. Thanks to this new protocol, all O. oeni strains used in the study were typed using the five tandem repeats (TR). As expected, the primers for the five TR loci were specific to O. oeni. The method was improved to analyze isolated and mixed colonies, as well as bacteria harvested from wine using fast technology for analysis of nucleic acids (FTA®) technology. Finally, predictive models were constructed, to predict phylogenetic relationships and associate bacterial strain resistance to freeze-drying with fragment length analysis (FLA) profiles and genotypic and phenotypic characters.

Splitting of a Prevalent Mycobacterium bovis Spoligotype by Variable-Number Tandem-Repeat Typing Reveals High Heterogeneity in an Evolving Clonal Group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Application of variable-number tandem-repeat typing to discriminate Ralstonia solanacearum strains associated with English watercourses and disease outbreaks.

Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.

Clustering of Tuberculosis Cases Based on Variable-Number Tandem-Repeat Typing in Relation to the Population Structure of Mycobacterium tuberculosis in the Netherlands

The population structure of 3,776 Mycobacterium tuberculosis isolates was determined using variable-number tandem-repeat (VNTR) typing. The degree of clonality was so high that a more relaxed definition of clustering cannot be applied. Among recent immigrants with non-Euro-American isolates, transmission is overestimated if based on identical VNTR patterns.

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Multiple locus variable number tandem repeat analysis of Mycoplasma bovis isolated from local and imported cattle

Mycoplasma bovis is an important and emerging pathogen of cattle. In this study, multiple locus variable number tandem repeat (VNTR) analysis was used to differentiate M. bovis type strain PG45 and 68 M. bovis field isolates, including 34 isolates from calves imported to Israel from Australia, Lithuania and Hungary in the period 2006–2011, 32 isolates from mastitic dairy cows in Israel in the period 2000–2011, one isolate from the pneumonic lungs of a calf in Israel in 2010 and one isolate from frozen bull semen in Israel in 2008. A total of 35 VNTR types were distinguished, including three, eight and 10 different VNTR types among isolates from calves imported from Australia, Hungary and Lithuania, respectively, and 17 VNTR types among isolates from dairy cows in Israel. The VNTR types in isolates from Lithuanian calves were not identified among isolates from Israeli dairy cows. VNTR type XX, present in the Hungarian group, was identified in one Israeli mastitis-associated isolate. A cluster of 16 M. bovis isolates from Israeli dairy cows possessed the same VNTR type III as three Australian isolates from a single shipment of calves in 2006. The other cluster of isolates contained M. bovis strain 883, isolated from a mastitic cow, strain 72236, isolated from a calf with pneumonia, two isolates from calves imported from Australia to the same farm 3months previously and four isolates from calves in quarantine imported to Israel from Australia in 2009–2010. Multiple locus VNTR analysis is a useful tool for understanding the movement and spread of strains of M. bovis within and across international boundaries.

Epidemic Spread of Multidrug-Resistant Tuberculosis in Johannesburg, South Africa

Numerous reports have documented isolated transmission events or clonal outbreaks of multidrug-resistant Mycobacterium tuberculosis strains, but knowledge of their epidemic spread remains limited. In this study, we evaluated drug resistance, strain diversity, and clustering rates in patients diagnosed with multidrug-resistant (MDR) tuberculosis (TB) at the National Health Laboratory Service (NHLS) Central TB Laboratory in Johannesburg, South Africa, between March 2004 and December 2007. Phenotypic drug susceptibility testing was done using the indirect proportion method, while each isolate was genotyped using a combination of spoligotyping and 12-MIRU typing (12-locus multiple interspersed repetitive unit typing). Isolates from 434 MDR-TB patients were evaluated, of which 238 (54.8%) were resistant to four first-line drugs (isoniazid, rifampin, ethambutol, and streptomycin). Spoligotyping identified 56 different strains and 28 clusters of variable size (2 to 71 cases per cluster) with a clustering rate of 87.1%. Ten clusters included 337 (77.6%) of all cases, with strains of the Beijing genotype being most prevalent (16.4%). Combined analysis of spoligotyping and 12-MIRU typing increased the discriminatory power (Hunter Gaston discriminatory index [HGDI] = 0.962) and reduced the clustering rate to 66.8%. Resolution of Beijing genotype strains was further enhanced with the 24-MIRU-VNTR (variable-number tandem repeat) typing method by identifying 15 subclusters and 19 unique strains from twelve 12-MIRU clusters. High levels of clustering among a variety of strains suggest a true epidemic spread of MDR-TB in the study setting, emphasizing the urgency of early diagnosis and effective treatment to reduce transmission within this community.

Polymerase Chain Reaction-Free Variable-Number Tandem Repeat Typing Using Gold Nanoparticle–DNA Monoconjugates

In this work, we report a novel polymerase chain reaction (PCR)-free variable-number tandem repeat (VNTR) typing method using a T-shaped gold nanoparticle-DNA monoconjugate, called the "watching-gene assay". The T-shaped DNA probe was synthesized by "click" chemistry and linked with the gold nanoparticle to form the gold nanoparticle-DNA monoconjugate (a VNTR probe). Through a simple annealing and ligation reaction of the VNTR probe on a synthetic DNA template mimicking the human D1S80 VNTR locus, the number of tandem repeat units could be deciphered by counting the self-assembled gold nanoparticles. The number of tandem repeat units could be identified with more than 50% yield if the repeat number was less than four. In the case of the real human genomic DNA, the 18 repeat unit number could be successfully revealed by observing the 18-gold-nanoparticle cluster, which exactly corresponded to the number of tandem repeats of the real sample. Our "watching-gene assay" is rapid, simple, and direct for data interpretation, thereby providing an advanced PCR-free genetic polymorphism analysis platform.