Gene typing of infectious tularemia strains isolated from the Stavropol and Krasnodar Territories

The typing of F. tularensis strains by four variable number of tandem repeats (VNTR) loci has been carried out. Among the strains isolated in the Stavropol and Krasnodar Territories seven genotypes have been detected and their spread in different natural foci has been analyzed. The data thus obtained suggest that the VNTR analysis may become an important instrument for studying the structure of the natural foci of tularemia and evolutionary relationships between individual areas of these foci.     

Prenatal determination of a variable number of tandem repeats in intron 40 of the von Willebrand factor gene from maternal peripheral blood using the polymerase chain reaction

To determine a fetal variable number of tandem repeats (VNTR) without using fetal tissues, we amplified the whole VNTR region in intron 40 of the von Willebrand factor gene from maternal peripheral blood by the polymerase chain reaction and then separated the amplified fragments by a nonhydratable polyacrylamide gel with an electrolyte gradient. After water elution from the gel, each preliminary product was amplified for the second time and then digested by Alu I restriction endonuclease. Then the VNTR was examined using a hydratable polyacrylamide gel with an electrolyte gradient. We successfully identified the fetal VNTR from 6 cases of 10 maternal peripheral blood samples and 2 cases of 6 maternal blood samples after parturition. This study might offer a theoretical basis for the noninvasive diagnosis of genetic disorders and identification of disputed paternity with common primers.     

Vibrio cholerae O139 isolated from humans and the water from open reservoirs: a comparative genotyping

The comparative study of the genomes of V. cholerae O139 isolated from humans and from water of surface reservoirs was carried out with the use of single- and double-primer polymerase chain reaction (PCR). The profiles of polymorphic DNA fragments obtained in this study made it possible to find out differences between groups of strains, as well as the individual features of some of them. The comparison of strains isolated from humans and from water in single-primer PCR revealed that they, in spite of the general similarity of their genomes, essentially differed, which was probably due to changes in the genome of this infective agent. Strains of aqueous origin lacked genes ctx and tcpA, which made them epidemiologically unimportant.     

Characterization of Vibrio cholerae O1 cultures isolated from environmental objects on the territory of the Russian Federation in 2002

The biological properties of 46 V. cholerae O1 eltor cultures isolated in 2002 from water environment on the territory of Russia are presented. All isolated vibrios proved to be typical in their cultural, morphological, biochemical and serological properties. The atypical character of some of them was mainly linked with their phage resistance. The appearance of vibrios, sensitive to bacteriophage ctx+ and containing gene tcp in the absence gene ctx, was noted. Multilocus VNTR typing made it possible to group the cultures under study in 34 genotypes. The presence of toxin coregulated pili was found to be directly related to locus VcB. The necessity of the systematic study of the pheno- and genotypes of the isolated cultures with the aim of epidemiological surveillance of this infection is emphasized.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

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.     

Typing of Mycobacterium tuberculosis cultures from Samara region by the variable number of tandem repeats

To type Mycobacterium tuberculosis (MT) strains by the variable number of tandem repeats (VNTR), 136 MT cultures, isolated in 5 main general therapeutic laboratories and penitentiary antituberculosis institutions of the Samara region, were studied. Gene typing revealed that the greatest number of MT strains (73) belonged to VNTR type 42435. It was followed by VNTR type 22232, found in 13 isolated cultures. Among the MT cultures of the most widely spread VNTR type 42435, rifampicin-resistant strains prevailed (57 strains, i.e. 78%), while strains belonging to VNTR type 22232 were mainly sensitive to rifampicin (84%). VNTR typing of MT typing may be useful for epidemiological studies in the field of phthisiology.     

Genetic relationships of Bacillus anthracis and closely related species based on variable-number tandem repeat analysis and BOX-PCR genomic fingerprinting

Variable-number tandem repeats (VNTR) analysis and BOX-repeat-based PCR (BOX-PCR) genomic fingerprinting were performed on 25 Bacillus strains to investigate the genetic relatedness of Bacillus anthracis to the closely related species. Based on VNTR analysis, all B. anthracis strains could be assigned to (VNTR)(4), which is the most commonly found type in the world. Interestingly, a (VNTR)(2) was also observed in Bacillus cereus KCTC 1661 and with an exact match to the tandem repeats found in B. anthracis. This finding has never been reported before in the closely related species. According to the BOX-PCR, B. anthracis strains clustered together and separated reliably from the closely related species. However, B. cereus KCTC 1661 was linked to the B. anthracis cluster and showed close relationships with B. anthracis strains. These results indicated that there was a strong correlation between VNTR analysis and BOX-PCR genomic fingerprinting.     

Differentiation of strains of Xylella fastidiosa by a variable number of tandem repeat analysis

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.     

Association analysis of IL-4 VNTR polymorphism with rheumatoid arthritis in Iranian patients

Rheumatoid arthritis (RA) is an autoimmune disease characterized by movement disability and pain in the joints. The affected individuals are susceptible to other subsequent diseases, exacerbating the condition. To find out the genetic variability of this disease at the genomic level for the first time in the Iranian population, we carried out an investigation on the VNTR of IL-4 gene within its third intron. For this goal we isolated the genomic DNA from blood samples of 576 rheumatoid arthritis patients and 546 healthy controls and investigated the presence or absence of specific amplicons via polymerase chain reaction (PCR). The size of each amplicon on a 1.5% agarose gel corresponded to a certain number of tandem repeats which indicated a specific allele. Statistical test of χ² Fisher’s exact test and odds ratio (OR) was used to analyze the data. The results showed that RA1/RA1 genotype was the dominant genotype in both healthy controls and patients and the heterozygote genotype of RA1/RA2 was observed more in the healthy controls than patients (108 vs. 66) with significant difference with P value < 0.005 and odds ratio of 0.214. However two genotypes of RA2/RA2 and RA2/RA3 were exclusively observed in the patients’ samples with P value = 0.023 and odds ratio of 0.988. We concluded that IL-4 VNTR polymorphism has a strong association with rheumatoid arthritis and might be a high risk factor for development of rheumatoid arthritis in the investigated Iranian population.     

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.     

Polymers of random short oligonucleotides detect polymorphic loci in the human genome.

Polymers of random 14 mer oligonucleotides are shown to detect discrete loci in the human genome. Eighteen different synthetic tandem repeats of random 14 base-pair units (STRs) have been generated and all of them turn out to detect polymorphic loci on southern blots of human DNA samples, presumably corresponding to a variable number of tandem repeats (VNTR). This finding suggests that minisatellites are a major component of the human genome and are strongly associated with the generation of genetic variability. In addition, it should open new strategies to make new polymorphic probes available.     

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.     

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.     

Differentiation of Strains of Xylella fastidiosa by a Variable Number of Tandem Repeat Analysis

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.     

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.     

Evolution of a VNTR located within the promoter region of the thiopurine methyltransferase gene: inferences from population and sequence data

The promoter region of the human thiopurine methyltransferase (TPMT) gene contains a variable number of tandem repeats (VNTR) with three kind of motifs (A, B, and C) differing by the length of the unit core and nucleotide sequence. We have studied the structural variation within the VNTR alleles in two human populations and in samples from gorillas and chimpanzees. In humans, no intermingling of motifs was detected within the VNTR, and the sequences of the three core motifs remained remarkably unchanged, differences between alleles corresponding essentially to variations in the number of A and B repeats. The variation pattern in humans is consistent with an interpretation in which two contiguous genetic units (repeats A and B) behave evolutionarily according to the stepwise mutation model, as inferred from the population distribution profiles and from the molecular phylogenetic relationships among the VNTR alleles. However, the observation of a strong negative correlation between the numbers of A and B repeats also suggests that the regularity and/or independence of the mutational process has been disrupted to some extent by interactions between the A and B stretches. Selective pressure (the VNTR plays some role, although minor, in the TPMT function) or biased mutation are possible explanations. In gorillas and chimpanzees, several A-, B-, or C-like motifs were detected, but their arrangement within the VNTR alleles did not followed the regular pattern registered in humans and, particularly for the B-like motifs, a considerable sequence hypervariability was registered. Furthermore, the structural differences among non-human alleles were sufficiently numerous to render more plausible the assumption of the infinite allele model.     

Multiple-locus variable-number tandem repeat analysis of Salmonella enterica subsp. enterica serovar Typhimurium: comparison of isolates from pigs, poultry and cases of human gastroenteritis

AIMS: Pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) profiles of 195 epidemiologically unrelated Salmonella Typhimurium strains isolated in 1997-2004 from pigs were analysed and the results compared to establish the discriminatory ability of each method. In order to investigate the epidemiology of S. Typhimurium from different populations, the VNTR profiles from pigs were compared with those obtained from 190 S. Typhimurium strains isolated from poultry and 186 strains isolated from human cases of gastroenteritis. METHODS AND RESULTS: A total of 195 strains of S. Typhimurium were tested by PFGE and VNTR. For PFGE, the restriction enzyme XbaI was used, and for VNTR, the number of repeats at five loci (STTR 9, 5, 6, 10pl and 3) were counted and assigned an allele number based on an established VNTR scheme. The results obtained showed improved discrimination of VNTR when compared with PFGE with 34 PFGE profiles identified compared with 96 different VNTR profiles for the pig isolates and 56 different VNTR types within the most common PFGE type. Within the three different populations, VNTR showed distinct subpopulations of VNTR type related not only to source, but also demonstrated common VNTR types within samples obtained from humans, poultry and pigs, especially in strains of phage type DT104. CONCLUSIONS: VNTR has taken the discrimination to a further level than that obtained through PFGE, and demonstrated an overlap in the genetic diversity of isolates tested across the three different populations, confirming previous suggestions that animals have an involvement in the dissemination of S. Typhimurium through the food chain. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella Typhimurium remains an important concern as a food-borne zoonotic agent. The VNTR strategy described provides an accurate method of tracing strain dissemination, and adds a further level of discrimination to the PFGE type, providing potential benefits to epidemiological studies and the possibility of deciphering source attribution of cases.     

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.     

PCR analysis of four length-polymorphic loci in Korean population for genotyping

On human chromosomes, a short sequence of DNA is known to repeat a number of times. These repeats are called variable number of tandem repeat (VNTR) or short tandem repeat (STR) which has a short repeat core. VNTR and STR are used in the field of forensic science, evolution, and anthropology. In this work, we examined allele frequencies of one VNTR (YNZ22) and three STRs (NeuR, D21S11, Humth01) in a Korean population sample by polymerase chain reaction (PCR) followed by high-resolution polyacrylamide gel electrophoresis (PAGE) with silver stain. Subsequently, the polymorphism information content (PIC) was calculated: the highest PIC was observed in the NeuR locus (0.95680) and lowest in the Humth01 locus (0.75809).