Evaluation of VNTR typing for the identification of Mycobacterium ulcerans in environmental samples from Victoria, Australia

Reliable molecular detection of Mycobacterium ulcerans in environmental samples is essential to study the ecology and transmission of this important human pathogen. Variable number tandem repeat (VNTR) typing is a valuable method for distinguishing M. ulcerans isolates from different geographic regions and for distinguishing M. ulcerans from other members of the Mycobacterium marinum/M. ulcerans complex, but its application to environmental samples has not yet been evaluated systematically. This study compares the sensitivity and specificity of PCR detection of 13 VNTR loci to determine the best loci for the analysis of environmental samples. This study demonstrates that VNTR typing using selected loci can be a useful addition to established molecular methods for detecting M. ulcerans in the environment and highlights some of the issues encountered when using molecular methods to detect microorganisms in environmental samples. When applied to environmental samples collected from an endemic region in Victoria, Australia, VNTR typing confirmed that the strain of M. ulcerans being detected was indistinguishable from the strain causing disease in humans in that region.     

High resolution melting analysis for detection of variable number of tandem repeats polymorphism of XRCC5

Several polymorphisms in the XRCC5 (X-ray repair cross-complementing 5; OMIM: 194364) were reported. Polymorphism of variable number of tandem repeats (VNTR) in the promoter region of XRCC5 (rs6147172) was reported. The main aim of the present study is to introduce the high resolution melting analysis (HRMA) method for genotyping of the polymorphism of XRCC5 VNTR. Genotypes of XRCC5 VNTR were determined by HRMA and conventional PCR method, and confirmed by DNA sequencing. The results for genotyping using HRMA and conventional PCR showed 100% concordance. All genotypes of the XRCC5 VNTR polymorphism could be accurately detected by HRMA.     

Evaluation of three molecular methods of repetitive element loci for differentiation of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> (MAP)

The aim of the present study is to evaluate the efficiency of three methods to determine the molecular diversity of 34 Mycobacterium avium subsp. paratuberculosis (MAP) strains isolated from 17 cattle herds. The applied methods included the analysis of sequence polymorphism of the mononucleotide (G1 and G2) and trinucleotide sequences (GGT) of the Short Sequence Repeats (SSR) and the determination of size polymorphism of 9 different Mycobacterial Interspersed Repetitive Units (MIRU) and 6 Variable Number Tandem Repeats (VNTR). Sequence analysis of SSR of 34 isolates showed 4, 6, and 2 alleles of G1, G2, and GGT repeats, respectively. The amplification of the investigated 9 MIRU units revealed only two discriminatory genotyping systems (MIRU2 and MIRU3). Out of 6 VNTR PCR differentiation methods, only one method could be recommended for genotyping purposes. The profile 7g-12g-4ggt-II-b-2 of the combination systems G1-G2-GGT-MIRU2-MIRU3-VNTR1658 dominates among the examined isolates and was detected in 14.7% of the isolates. The use of certain repetitive loci of SSR, MIRU, and VNTR techniques in this study showed greater potential than others for the characterization of MAP isolates. The recommended loci can be used for the epidemiological tracing of MAP field strains and to determine the relationships between isolates in different herds.     

Application of MIRU–VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU–VNTR). According to multiple studies, MIRU–VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU–VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU–VNTR on smear slides in detecting mixed infections. Ziehl–Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU–VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU–VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU–VNTR on smear slides can be conveniently used for the detection of mixed infections.

     

Characterization of three VNTR systems at D21S112.

D21S112 is a highly polymorphic marker on the long arm of chromosome 21. Our analysis of this locus indicated the presence of three VNTR systems. We estimated the heterozygosity of each system and sequenced one of the repetitive regions. Utilizing PCR, we demonstrated that the sequenced VNTR is responsible for the system with the highest level of heterozygosity. Combining data from the three systems makes D21S112 one of the most informative loci on the chromosome.     

Molecular-genetic analysis of VNTR polymorphism in alleles of the phenylalanine hydroxylase gene in inhabitants of the Volga-Urals region

Molecular genetic analysis of the VNTR alleles at the phenylalanine hydroxylase (PAH) gene was carried out in seven Volga-Ural ethnic groups (Bashkirs, Tatars, Chuvashes, Maris, Mordovians, Udmurts, and Komis). The PCR fragments revealed included alleles of 380, 440, 470, 500, 530, 560, and 650 bp, containing 3, 5, 6, 7, 8, 9, and 12 repeat copies, respectively. Substantial heterogeneity of the populations in respect to the distribution and frequency of the VNTR alleles and genotypes was demonstrated. The indices of observed and theoretical heterozygosity of the PAH VNTR alleles were calculated. The mean heterozygosity index was 70.02%. This high index value along with the established differentiation of the populations in respect to the frequency distribution of the VNTR alleles and PAH genotypes permitted the conclusion that the given polymorphic locus can serve as a highly informative marker for examination of the genetic structure of the Volga-Ural populations.     

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.     

High-throughput typing method to identify a non-outbreak-involved Legionella pneumophila strain colonizing the entire water supply system in the town of Rennes, France

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.     

The use of MLVA for Corynebacterium diphtheriae genotyping--preliminary studies

The complete genome sequence of strain NCTC 13129 C. diphtheriae were investigated in order to identify tandem repeats (VNTR). From 75 VNTR loci identified in the genome 14 were selected. Primers were designed and PCR conditions were optimized for amplification of the selected VNTR markers. Preliminary studies of usefulness of selected VNTR markers were conducted using a group of 28 C. diphtheriae strains. From 14 markers 8 were regarded as potentially useful. The diversity of individual markers ranged from 1 to 6 alleles (Simpson index from 0 to 0,746). No diversity were observed for 3 VNTR markers but it could be a results of too small group of strains analyzed in the tests. Simpson diversity index calculated for all the markers tested on 28 strains was 0,87. Results of the preliminary studies showed usefulness of MLVA for C. diphtheriae genotyping. Nevertheless, confirmation of reliability of the method should be done using a large group of strains. Moreover, the method should be compared with other genotyping methods.     

Molecular Techniques in the Assessment of Botanical Diversity

A variety of different molecular techniques can be used forthe study of botanical diversity. Restriction fragment lengthpolymorphism (RFLP), arbitrary primed DNA, amplified fragmentlength polymorphism (AFLP), variable number of tandem repeats(VNTR), sequence-tagged simple sequence repeats (SSRs) and polymerasechain reaction (PCR) sequencing are briefly reviewed here. Thesetechniques differ in the way that they resolve genetic differences,in the type of data that they generate and in the taxonomiclevels at which they may be most appropriately applied. It isimperative to understand the different ways in which the datafrom the different molecular techniques can be utilized beforeembarking upon a programme of applying them to any particulardiversity study. Biodiversity; RFLP; RAPD; AFLP; VNTR; SSR; molecular data; fingerprinting; genetic distances; phylogeny; conservation     

The VNTR polymorphism in the human dopamine transporter gene: improved detection and absence of association of VNTR alleles with attention-deficit hyperactivity disorder

The human dopamine transporter (DAT1) gene contains a variable number tandem repeat (VNTR) in its 3'-untranslated region because of repetition of a 40-bp core sequence. Methods available for the diagnosis of this polymorphism are limited in number. We have developed a new polymerase chain reaction (PCR) test, which is similar to that described originally by Vandenbergh's group, but provides a better detection of the VNTR alleles in the human DAT1 gene. Using two independent PCR methods, we have determined the distribution of VNTR alleles in 110 healthy Omani subjects, and in 92 children with attention-deficit hyperactivity disorder (ADHD). The frequency of the risk allele (DAT1*10) was similar in the healthy subjects and ADHD cases, indicating absence of association of this allele with ADHD in Oman.     

Mutations in the VNTR of the carboxyl-ester lipase gene (CEL) are a rare cause of monogenic diabetes

We have previously shown that heterozygous single-base deletions in the carboxyl-ester lipase (CEL) gene cause exocrine and endocrine pancreatic dysfunction in two multigenerational families. These deletions were found in the first and fourth repeats of a variable number of tandem repeats (VNTR), which has proven challenging to sequence due to high GC-content and considerable length variation. We have therefore developed a screening method consisting of a multiplex PCR followed by fragment analysis. The method detected putative disease-causing insertions and deletions in the proximal repeats of the VNTR, and determined the VNTR-length of each allele. When blindly testing 56 members of the two families with known single-base deletions in the CEL VNTR, the method correctly assessed the mutation carriers. Screening of 241 probands from suspected maturity-onset diabetes of the young (MODY) families negative for mutations in known MODY genes (95 individuals from Denmark and 146 individuals from UK) revealed no deletions in the proximal repeats of the CEL VNTR. However, we found one Danish patient with a short, novel CEL allele containing only three VNTR repeats (normal range 7–23 in healthy controls). This allele co-segregated with diabetes or impaired glucose tolerance in the patient’s family as six of seven mutation carriers were affected. We also identified individuals who had three copies of a complete CEL VNTR. In conclusion, the CEL gene is highly polymorphic, but mutations in CEL are likely to be a rare cause of monogenic diabetes.     

Highly sensitive chemiluminescent method for the detection of cell contamination

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination. © 1998 John Wiley & Sons, Ltd.     

Association of variable number of tandem repeats polymorphism in the IL-4 gene with end-stage renal disease in Malaysian patients

Variable number of tandem repeats (VNTR) polymorphism in the interleukin 4 (IL-4) gene has been associated with end-stage renal disease (ESRD) subjects in many different populations, although with conflicting results. We determined the 70 bp of VNTR polymorphism at intron 3 of the IL-4 gene in Malaysian ESRD subjects. Buccal cells were collected from 160 case and 160 control subjects; genomic DNA was amplified using PCR, followed by agarose gel electrophoresis. There were significant differences in genotypes and alleles of the IL-4 gene. We conclude that VNTR polymorphism of the IL-4 gene is a risk factor for the development of ESRD among Malaysians.     

Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies

The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman^® technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs’ distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.     

Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.     

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii

The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.     

Variable nucleotide tandem repeats (VNTR analysis) in Vibrio cholerae 0139 isolated from humans and from water of surface reservoirs in Russia

The comparative study of variable tandem repeats (VNTR analysis) in genomes of V. cholerae 0139 isolated from humans and from water samples taken from surface reservoirs was carried out. The results of the study of the allele state of 5 loci of tandem repeats in 50 strains of vibrios, carried out in the double-primer polymerase chain reaction (PCR), as well as the earlier comparison of the same isolates in the single-primer PCR, showed essential differences and the absence of clonality in the cultures of the clinical and aqueous origin. The suggestion was made that vibrios with individual VNTR genotypes and having no genes ctx and tcpA, isolated from water samples, were epidemic unimportant representatives of the autochthonous microflora of water reservoirs.     

用DNA多态性分析福建,江西和浙江3省畲族的亲缘关系

为分析福建,江西和浙江３省畲族之间的关系,用聚合酶链反应（ＰＣＲ）对上述３省的３代均为畲族的无关个体的载脂蛋白Ｂ基因和Ｄ１７Ｓ３０位点的数目可变的串联重复（ＶＮＴＲ）序列进行研究。结果为：６个共有的ａｐｏＢ ＶＮＴＲ和５个共有的Ｄ１７Ｓ３０ ＶＮＴＲ等位基因在３省畲族中的分布相同,为进一步在基因水下上研究它们的亲缘关系奠定了基础。本文报道的检测ＤＮＡ多态性的方法在研究民族起源,变迁和民族识别以及各     

New approach for isolation of VNTR markers.

Elsewhere we have reported an efficient method for isolating VNTR (Variable Number of Tandem Repeats) markers. Several of the VNTR markers isolated in those experiments were sequenced, and a DNA sequence of 9 bp (GNNGTGGG) emerged as an apparent consensus sequence for VNTR markers. To confirm this result and to develop more VNTR markers, we synthesized nine different 18-base-long oligonucleotides whose sequences each included GNNGTGGG. When 102 cosmid clones selected by these oligonucleotides were tested for polymorphism, 34 (33%) of them showed multiallelic VNTR polymorphisms (average heterozygosity 68%). This procedure represents a new and efficient approach for isolating additional VNTR markers and supports the idea that the GNNGTGGG sequence may play an important role in the generation of the multiallelic systems within the human genome.