Restriction Fragment Length Polymorphism Typing Research Articles

High resolution genetic diversity studies of marine Synechococcus isolates using rpoC1-based restriction fragment length polymorphism

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 45:263-275 (2006) - doi:10.3354/ame045263 High resolution genetic diversity studies of marine Synechococcus isolates using rpoC1-based restriction fragment length polymorphism Martin Mühling1,2,*, Nick J. Fuller2, Paul J. Somerfield1, Anton F. Post3, William H. Wilson1, David J. Scanlan2, Ian Joint1, Nicholas H. Mann2 1Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, Devon, UK 2Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, Midlands, UK 3InterUniversity Institute for Marine Science, Coral Beach, 88103 Eilat, Israel *Email: mmuh@pml.ac.uk ABSTRACT: Synechococcus-specific polymerase chain reaction (PCR) primers were developed and used to amplify fragments of the RNA polymerase core subunit gene rpoC1 of a set of 23 marine isolates from different oceanographic locations. Restriction fragment length polymorphism (RFLP) analysis of the PCR products differentiated between most strains and all phylogenetic clades, thus allowing the screening of large sample sets at high genetic resolution. The method was used to analyse changes in the genetic diversity of marine Synechococcus strains through an annual cycle at 100 m depth in the Gulf of Aqaba, Red Sea, and along the depth profile of a sample from June 1999. RFLP analysis of 36 clones of each of the 8 rpoC1 clone libraries screened showed that the Synechococcus population during summer and autumn was dominated by 1 to 3 genetically different clones, while maximum genetic richness was found during spring and winter. Analysis of the depth profile during the period of stratification revealed differences in relative abundance of particular RFLP types among depths (10, 30, 100 m), although the same genotypes dominated the assemblage throughout. Multivariate analysis of environmental variables and Synechococcus assemblage composition showed that Synechococcus genetic diversity was most closely, and significantly, correlated to a combination of temperature and NO3– (ρ = 0.645, p = 0.038). Phylogenetic analysis of the marine rpoC1 nucleotide sequences of the 40 different RFLP types identified in this study, and all Synechococcus rpoC1 sequences in the databases, demonstrates that the new PCR primers amplify rpoC1 gene fragments from all MC-A and some Cyanobium lineages of marine Synechococcus strains and are also useful for the identification of potentially novel lineages. KEY WORDS: Synechococcus spp. · Marine · rpoC1 · Genetic diversity · Population dynamics Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 45, No. 3. Online publication date: December 21, 2006 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2006 Inter-Research.

Restriction Fragment Length Polymorphism Typing of Infectious Bursal Disease Virus Field Strains in Turkey

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions.

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing ofMycobacterium tuberculosis

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).

SUBSPECIES CHARACTERIZATION OF UREASE-POSITIVE THERMOPHILIC CAMPYLOBACTER (UPTC) ISOLATED FROM SHELLFISH EMPLOYING MODIFIED FLAGELLIN (flaA) RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) TYPING

Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction—restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.

An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine

Ochratoxigenic mycobiota in grapes from representative wine regions in Valencia was identified. Black aspergilli were predominant among the different Aspergillus spp. isolated. Restriction digestion analysis of the ITS products was tested as a rapid method to identify isolates of black Aspergillus species from grapes. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI, distinguished five types of restriction fragment length polymorphism (RFLP) corresponding to Aspergillus niger, Aspergillus tubingensis, Aspergillus carbonarius and Aspergillus aculeatus species. In addition, a new RFLP type in the A. niger aggregate was identified. The fragments obtained by digestion with the endonuclease NlaIII could be used to identify these new isolates. Black Aspergillus isolates were tested for their ability to produce OTA. Most of the isolates that produced ochratoxin A in YES medium belonged to A. carbonarius species. These results support evidence that A. carbonarious greatly contributes to OTA contamination in grapes and consequently in wine. The ITS-RFLP assay is proposed as a rapid and easy method to identify black Aspergillus species isolated from grapes, especially in studies that involve a large number of isolates.

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia

The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated. Aspergillus niger and A. carbonarius were isolated from vineyard soils. Aspergillus niger was more widespread than A. carbonarius, and two restriction fragment length polymorphism types of A. niger, N and T, were present. Three of 113 A. niger isolates and all 33 A. carbonarius isolates produced OA. Aspergillus carbonarius was inoculated onto Semillon bunches with and without damage in the month before harvest. Damaged berries at greater than 12.3 (o) Bx were particularly susceptible to Aspergillus rot and production of OA, which was concentrated in severely mouldy berries. OA in Australian grapes results mainly from infection of berries by A. carbonarius. It is concentrated in discoloured, shrivelled berries. The potential for Aspergillus rot and OA production appears to commence after veraison and increase with berry damage and ripeness. Minimizing damage to grapes between veraison and harvest significantly reduces Aspergillus rot and OA formation. Monitoring the extent of Aspergillus rot in bunches infected with toxigenic Aspergillus spp. may give some indication of OA contamination.

Detection of Mycobacterium avium subsp. paratuberculosis in Two Brown Bears in the Central European Carpathians

The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-C1, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection.

Relationship of trichloroethylene-induced medicamentosa like dermatitis to HLA-DMA and HLA-DMB

To investigate the susceptibility of trichloroethylene-induced medicamentosa like dermatitis by comparing the frequency of HLA-DMA and HLA-DMB in patients with trichloroethylene-induced medicamentosa like dermatitis and in normal controls. The DNA of lymphocytes in 61 patients with trichloroethylene-induced medicamentosa like dermatitis and in 60 people as the normal control were abstracted by using touchdown PCR amplification of HLA-DMA and HLA-DMB. Then through restriction fragment length polymorphism (RFLP) and sequence base typing, the alleles and genotypes were confirmed. The frequency of HLA-DMA and HLA-DMB in the two groups was compared. The HLA-DMA*0101 allele frequency in patients with trichloroethylene-induced medicamentosa like dermatitis was significantly higher than in the control group (71.3% vs 55.0%, P < 0.05). The allele frequency of HLA-DMA*0103 was significantly higher in the patient group than in the control group (11.5% vs 3.3%, P < 0.05). The ratio of *0102 homozygotes of HLA-DMA*0102 in the patient group was significantly higher than in the control group (25.0% vs 8.2%, P < 0.05). The ratio of *0102 heterozygotes of HLA-DMB*0101 in the patient group was lower than in the control group (P < 0.05). The polymorphisms of DMA may be related to the susceptibility of the patients with trichloroethylene-induced medicamentosa like dermatitis.

Soluble di‐iron monooxygenase gene diversity in soils, sediments and ethene enrichments

Soluble di-iron monooxygenases (SDIMOs) are key enzymes in the bacterial oxidation of hydrocarbons, and have applications in environmental and industrial biotechnology. SDIMOs from pure cultures are unlikely to represent the total diversity of this enzyme family, so we used polymerase chain reaction to survey the diversity of SDIMO alpha subunit genes in environmental samples, ethene enrichments and ethene-degrading bacterial isolates. From 178 cloned amplicons, 98 restriction fragment length polymorphism types were seen, from which 75 representative SDIMO sequences were obtained; 45 from environmental samples, 25 from enrichments and seven from isolates. The sequences were diverse, including genes similar to ethene (etnC), propene (amoC, pmoC), propane (prmA) and butane (bmoX) monooxygenases, in addition to many novel sequences comprising a new SDIMO group (group 6). Environmental samples showed the highest diversity, with strong representation of group 6 SDIMOs and prmA-like genes. Ethene stimulation of samples resulted in increased frequencies of group 4 SDIMOs (etnC-like). Four ethene-utilizing Mycobacterium isolates (NBB1-NBB4) from enrichments all contained etnC; one isolate (NBB4) also contained three additional SDIMO genes (bmoX-like, amoC-like and group 6). The primers, database, clone libraries and strains reported here provide a resource for future bioremediation and biocatalysis studies, with particular relevance for chlorinated alkene and alkane compounds.

Enrichment for microbes living in association with plant tissues

To investigate a cultivation-independent method of enrichment for microbes living in association with plant tissues. A large quantity of leaves or seeds was enzymatically hydrolyzed, and the pellets were collected by differential centrifugation. Enzyme concentration, buffer and incubation time were optimized for release of plant-associated microbes. The relative abundance of plant nuclear DNA and bacterial DNA in the enriched sample was estimated by PCR amplification of genome-specific marker genes. The efficiency of microbe enrichment was estimated from the proportion of bacterium-derived clones and their restriction fragment length polymorphism (RFLP) types as detected by 16S rRNA gene-based techniques. With a higher ratio of bacterial to plant nuclear DNA, the enriched samples showed a considerably enhanced proportion of bacterium-derived clones and a wider sequence diversity of those clones. The method described here proved to be remarkably effective in enriching for bacteria living in association with plant tissues. The method can be applied to study plant-associated microbes in the field of environmental molecular ecology and environmental metagenomics.

Homogeneity of Borrelia japonica and heterogeneity of Borrelia afzelii and 'Borrelia tanukii' isolated in Japan, determined from ospC gene sequences.

Borrelia afzelii, B. japonica, and 'B. tanukii' isolated from various sources and geographical origins in Japan were characterized by restriction fragment length polymorphism (RFLP) analysis and sequencing analysis of the outer surface protein C (OspC) amplicon. B. afzelii and 'B. tanukii' generated variable RFLP patterns and differences in ospC gene sequence were confirmed. In contrast, 26 isolates of B. japonica generated one OspC RFLP type, and sequence similarity between B. japonica ranged from 96.4 to 99.7%. These finding suggests that B. japonica is unique in comparison with other members of B. burgdorferi sensu lato species with respect to homogeneity of the ospC gene.

New Route of Importation ofMycobacterium tuberculosisBeijing Genotype

New Route of Importation of<i>Mycobacterium tuberculosis</i>Beijing Genotype

PCR-Based Restriction Fragment Length Polymorphism (RFLP) Analysis of Campylobacter jejuni Isolates from Humans, Chickens and Dogs in Northern Taiwan

Two hundred and twenty strains of Campylobacter jejuni (70 human, 51 canine and 99 chicken strains) were isolated from September 2003 to September 2004 in northern Taiwan. These strains were subtyped by PCR-RFLP analysis of the flagellin (FlaA) gene. On the basis of restrictive digest, six types were identified with AfaI, seven types with MboI and five types with HaeIII. With the combination of these three enzymes, 47 distinct PCR-RFLP patterns were observed-25 each from human and chicken isolates, and 9 from canine isolates. In human strains, the most frequently occurring types were Cj-28 (14.3%), Cj-17 (10%), Cj-16 (8.6%), Cj-37 (7.1%) and Cj-46 (7.1%). In canine strains, the most prevalent types were Cj-1 (33.3%), Cj-26 (19.6%), Cj-3 (15.7%), Cj-2 (9.8%) and Cj-10 (9.8%). In chicken strains, the most frequently occurring types were Cj-46 (40.4%), Cj-29 (9.1%), Cj-45 (7.1%) and Cj-41 (5.1%). The results suggest that poultry is a source, but not the sole source, of C. jejuni infection in humans. Two RFLP types, Cj-17 and Cj-37, frequently occurring in human isolates in this study have also been found to be prevalent in human isolates in Japan, China and the Czech Republic, indicating a possible international clonal spread.

Determination of Porcine Rotavirus Serotypes by RT-PCR and RFLP Analysis

G and P tying of group A porcine rotaviruses (PoRV) from field fecal samples were performed using reversetranscriptase polymerization chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis. After amplifying full length VP7 and partial length VP4 genes, restriction endonucleases were used to digest and analyze the cutting pattern of the gene products. After analysis of digests with restriction endonucleases, seven and six RFLP types were observed for VP7 and VP4, respectively. The G typing analysis of 50 fecal samples revealed that 68% (34/50) were G4, which included G4-like (22/50); 22% (11/50) were G5; 6% (3/50) were G4 and G5 mixed types. The P typing analysis of the same fecal samples revealed that 36% (18/50) were P2B, 52% (26/50) were P9, 1 sample (2%) was a mixture of P2B and P9. Combinations of G and P types, the G4P2B and G4P9 types including G4-like accounted for 26% (13/50) and 32% (16/50), respectively. The G5P2B and G5P9 type also represented 4% (2/50) and 18% (9/50) of the samples. No G3 and G11 or other new P types were identified from the samples tested. Information on the G and P types and G/P combinations in the field fecal samples is useful for developing more effective PoRV vaccines and understanding the epidemiology of PoRV infections in the field.

Evidence for local transmission and reactivation of tuberculosis in the Toronto Somali community

To further understand the molecular and clinical epidemiology of tuberculosis in the Toronto Somali community, molecular fingerprinting using IS6110 restriction fragment length polymorphism typing or spoligotyping was performed on M. tuberculosis isolates obtained from Somali-Canadians who developed active disease from 1997 to 2001. Molecular fingerprints were further compared with those obtained from Somalis residing in Denmark. 142 Somali TB patients were reported, for whom, 80 isolates were fingerprinted. 25% of isolates were clustered. Three clusters involving 2 patients each were identified out of the17 isolates that underwent spoligotyping. Of the 63 isolates typed by the IS6110 method, 6 clusters (4 of 2 patients and 2 of 3 patients) were identified. 57% of these isolates were found to be identical to Danish isolates. Our study suggests that a combination of reactivation and recent transmission are responsible for the high incidence rates of tuberculosis in this community. We recommend that ongoing surveillance and treatment programmes be directed towards this community.

Community analysis of arbuscular mycorrhizal fungi in a warm-temperate deciduous broad-leaved forest and introduction of the fungal community into the seedlings of indigenous woody plants

A community of arbuscular mycorrhizal (AM) fungi was investigated in a warm-temperate deciduous broad-leaved forest using a molecular analysis method. Root samples were obtained from the forest, and DNA was extracted from the samples. Partial 18S rDNA of AM fungi were amplified from the extracted DNA by polymerase chain reaction using a universal eukaryotic primer NS31 and an AM fungal-specific primer AM1. After cloning the PCR products, 394 clones were obtained in total, which were divided into five types by restriction fragment length polymorphism (RFLP) with HinfI, RsaI, and Hsp92II. More than 20% of the clones were randomly selected from each RFLP type and sequenced. Phylogenetic analysis showed that all the obtained clones belonged to Glomus but could not be identified at species level. Topsoil of the forest containing plant roots was inoculated to nonmycorrhizal seedlings of indigenous woody plants, Rhus javanica var. roxburghii and Clethra barvinervis, to introduce the community of AM fungi into the seedlings. Among these five RFLP types, four types were detected from both seedlings, which indicates that the AM fungal community in the forest root samples was introduced at least partly into the seedlings. Meanwhile, an additional four types that were not found in the forest root samples were newly detected in the seedlings, these types were closely related to one another and close to G. fasciculatum or G. intraradices. It is expected that a community of indigenous diverse AM fungi could be introduced into target fields by planting these mycorrhizal seedlings.

Potential risk of Mycobacterium avium subspecies paratuberculosis spread by syrphid flies in infected cattle farms

The syrphid Eristalis tenax Linnaeus (Diptera: Syrphidae) may be found in and around dung storage pits at cattle farms at various developmental stages of their life cycle. The purpose of this study was to investigate the occurrence of Mycobacterium avium ssp. paratuberculosis in 1044 E. tenax samples at various developmental stages, as well as fresh and stored dung originating from nine cattle farms. Mycobacterium fortuitum was isolated from one (1.5%) larva from the vicinity of three paratuberculosis-free herds of cattle. Mycobacterium a. paratuberculosis was isolated from 111 (21.4%) of E. tenax larvae collected from two of seven farms known to be infected with the causal agent of paratuberculosis. Mycobacteria were not isolated from any of the 340 pupae, 41 adults of 78 samples of exoskeletal exuviae. Mycobacterium a. paratuberculosis isolates from E. tenax larvae were of the IS900 restriction fragment length polymorphism (RFLP) type B-C1, identical to that detected in faecal samples from cattle herds infected with paratuberculosis. Larvae artificially infected with mycobacteria of IS900 RFLP type B-C9 did not contain statistically more CFU of identical IS900 RFLP type B-C9 in the intestinal tract and internal organs than on the body surface. These results show that M. a. paratuberculosis can survive in the intestinal tract and internal organs of E. tenax.

Efficient Differentiation of Mycobacterium tuberculosis Strains of the W-Beijing Family from Russia using Highly Polymorphic VNTR Loci

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen's University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter-Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a "gold standard" method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.