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PubMed2004

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae).

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

Animals↗

PubMed2002

Genetic variation in populations of Leishmania species in Brazil.

The population structure of strains of Leishmania (Viannia) braziliensis sensu lato from Pará State and Paraná State in Brazil, of L. (V.) shawi and L. (Leishmania) amazonensis from Pará State, and the relationships of type strains of the subgenera L. (Viannia) and L. (Leishmania) were examined by the random-amplified polymorphic deoxyribonucleic acid (RAPD) technique. Four different primers (M13-40, QG1, L15996 and delta gt11R) were used. The bands were analysed using the neighbor-joining (NJ) and unweighted pair-group method with arithmetic averages (UPGMA) algorithms of the MEGA package. The topology of the NJ and UPGMA trees was very similar but they were not always identical. Both trees differentiated the standard strains of the different species. Strains from the same location were grouped together only in the UPGMA phenogram of the M13-40 primer. L. (V.) braziliensis isolates from Paraná State were genetically closer to those from Paragominas, Pará State than to those from the Amazonian regions of Carajás in Pará State and Peru. The relationship was not dependent on geographical distance. It is postulated that the groups arose from different origins, in which the Amazonian stocks were related to Psychodopygus sand flies while the Paraná strains originated from a gene pool transmitted by Lutzomyia sand flies such as Lutzomyia (Nyssomyia) whitmani. Transmission by Ps. complexus in Paragominas is considered to be a secondary adaptation from the Lutzomyia leishmanial gene pool. Although the vectors of L. (V.) braziliensis are poorly known in the Amazon region, there is strong evidence that the major vectors are all Psychodopygus spp. There was a high degree of genetic variability amongst the L. (V.) shawi strains and there was no clear grouping according to the strains' origins. The genetic variability amongst L. (L.) amazonensis strains from the same locations was much lower but they formed 2 groups which coincided with their origin. Our results support the clonal population structure of Leishmania isolates and suggest that their distribution is related to the origin of the gene pool as well as to present vector and reservoir movements.

Animals↗

PubMed1999

Isolation and further characterization of phase variants of Streptococcus equi subsp. zooepidemicus.

In the present study the soft agar technique was used to isolate phase variants of S. equi subsp. zooepidemicus-cultures isolated from infections of horses. The phase variants were characterized by a compact or diffuse colony morphology in this media. The variants could be cultivated separately and further characterized genotypically by RAPD analysis and by macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis, indicating the identity of both strains of each pair. The diffuse colony variants grew uniformly turbid after cultivation in fluid media, did not haemagglutinate rabbit erythrocytes, and displayed a reduced surface hydrophobicity in hexadecane and phenyl-sepharose adherence tests. The compact colony variants generally grew as sediment with clear supernatant in fluid media, haemagglutinated rabbit erythrocytes and showed an enhanced surface hydrophobicity in both hydrophobicity tests. The presented soft agar technique allowed a demonstration of phase variation of S. equi subsp. zooepidemicus and a subsequent isolation of the variants. This might be an important prerequisite to understanding the pathogenic importance of phase variation among isolates of this bacterial species.

Animals↗

PubMed2005

Trypanosoma cruzi: two genetic groups in Paraná state, Southern Brazil.

This paper presents the genetic characterization of Trypanosoma cruzi strains isolated from chronic chagasic patients, triatomines, and sylvatic reservoirs from Paraná state, Southern Brazil, using the RAPD and SSR-PCR techniques. It has shown the presence of both phylogenetic groups of T. cruzi (I and II), describing for the first time the existence of T. cruzi II in Paraná state.

Animals↗

PubMed2002

Can composition and structural features of oligonucleotides contribute to their wide-scale applicability as random PCR primers in mapping bacterial genome diversity?

Among current genotypic methodologies, random amplification of polymorphic DNA (RAPD or AP-PCR) represents a widely employed assay for the evaluation of bacterial genomic diversity. A common bottleneck of this technique, however, is represented by the screening of useful informative primers to discriminate among isolates of a particular bacterial species. In an attempt to simplify this process, we evaluated here the utility of degenerate oligonucleotides to act as informative AP-PCR primers. For this purpose, a number of features (G+C contents, degeneracy rate, modifications at the 5' end) of related degenerate primers was tested for their effects in the generation of informative arrays from a set of bacterial genomes. Our results indicate that a combination of a wide base composition and a common palindromic structure at the 5' end of the sequences that compose the degenerate primers tested here beneficially resulted for the generation of informative arrays aimed to evaluate the bacterial genome heterogeneity.

5' Flanking Region↗

PubMed2002

Fruit plant germplasm characterisation using molecular markers generated in RAPD and ISSR-PCR.

The genotypes of the strawberry (Fragaria x ananassa), apple (Malus domestica) and Ribes species (R. nigrum, R. rubrum and R. glossularia), maintained in our Institute's collection and used in breeding programs, were screened for DNA markers. Twenty primers for RAPD (among 60 tested) and seven for ISSR (among 10 tested) were chosen as creating polymorphic DNA bands differentiating the investigated genotypes. Based on those identity markers, the genetic distance between genotypes was determined, and their relatedness was estimated. In many cases, both RAPD- and ISSR-based genetic similarity confirmed relatedness connected with biological origin and with the place where the cultivar was developed. However, some diversity connected with the technique used for molecular marker generation was observed. Generally, the similarity values based on ISSR data were higher than those based on RAPD. Parallel study using two data sets seems to enable a reduction in the number of potential mistakes connected with each method's, technical limitations and ensures more precise relatedness determination.

DNA Fingerprinting↗

PubMed2004

[Identification and mapping of chi115 gene and DNA markers linked to it in pea (Pisum sativum L.)].

Chlorophyll mutant Chi115 was induced by ethylmethane sulfonate (EMS) treatment of seeds of genotype Torsdag in Moscow State University and is characterized by lighter plant color. The monogenic nature of the mutant was determined by analyzing the F2 population from a cross between two P. sativum genotypes, WL1238 and Chi115. To establish a local map around the chi115 gene, the RAPD and ISSR techniques were used with 45 RAPD and 10 ISSR primers in combination with bulked segregant analysis (BSA). Linkage of 12 RAPDs and 2 ISSRs to the chi115 locus was observed in analysis of F2 single plants. Two RAPD markers that were closely associated with the chi115 gene were converted into the sequence characterized amplified region (SCAR) markers. By lowering the LOD score to 2, the linkage group containing the chi115 gene could be linked to the b gene (color of the flower) on linkage group III. Nevertheless, to prove the result obtained, three CAPS markers Sodmt, TubA1, and Rb were chosen on linkage group III. The results of linkage analysis showed that these CAPS markers were located within the linkage group including the chi115 gene.

Genes, Plant↗

PubMed2005

Molecular monitoring of spoilage yeasts during the production of candied fruit nougats to determine food contamination sources.

In the present work, we have analysed the yeast microbiota present in a manufacturing plant of candied fruits and nougats. Four yeasts species (Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Sporobolomyces roseus, and Debaryomyces hansenii) and a filamentous fungi (Nectria mauriiticola) were identified according to restriction analysis of 5.8S-ITS rDNA. These identifications were subsequently confirmed by sequencing the D1/D2 domain of the 26S rRNA gene. Z. rouxii and Z. bailii were isolated at high frequency along the whole manufacturing process. Since food alteration by Z. bailii and Z. rouxii is the cause of important economic losses for the food industry, there is a need for differentiating yeasts at the strain level as an essential part of quality control programs in this industry. For this purpose, we have tested the performance of three molecular techniques (RFLP mtDNA, RAPD-PCR, and microsatellite with (GAC)5 and (GTG)5 primers) to differentiate strains belonging to these two Zygosaccharomyces species. Those techniques with the best discriminatory power were applied to differentiate Zygosaccharomyces species isolates. The results of this analysis indicate that one strain of Z. bailii and two strains of Z. rouxii were involved in the spoilage of candied fruits. Moreover, the Z. bailii strain was also present in the spoiled nougat, hence being responsible of this alteration.

Candy↗

PubMed2004

Genotypic and phenotypic diversity of Lactobacillus delbrueckii subsp. lactis strains of dairy origin.

Eighty-nine strains of Lactobacillus delbrueckii subsp. lactis isolated from Italian hard and semi-hard cheeses and artisan starter cultures were characterised by phenotypic and genotypic methods. Phenotypic diversity was evaluated by studying biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR and pulsed field gel electrophoresis (PFGE). Phenotypic characterisation indicated a wide variability of the acidifying activity within Lact. delbrueckii subsp. lactis. Although the data was variable, it allowed us to evidence groups of strains with different acidifying properties, especially in terms of acidification intensity. Concerning peptidase activity, Lact. delbrueckii subsp. lactis showed a homogeneously high x-prolil-dipeptidil-aminopeptidase activity and a considerable but more heterogeneous lysil-aminopeptidase activity. The increased resolution obtained by the use of two molecular typing techniques, i.e. RAPD-PCR and PFGE, allowed to widen the level of strain heterogeneity. Technological and ecological pressures are determinant in selecting Lact. delbrueckii subsp. lactis sub-populations which are more functional to the different cheese technologies.

Cheese↗

PubMed2002

PCR based molecular technique for identification and discrimination of quarantined and non-quarantined Tilletia sps.

Polymerase chain reaction (PCR) based RAPD profiles, in conjunction with six primers, of Karnal bunt of wheat and rice bunt exhibiting distinct polymorphic DNA. A total of 84 RAPD loci were observed on polyacrylamide gel for both Tilletia sps. Out of 84, 16 loci were found monomorphic, while other 68 loci were unique. Usefulness of random primers was also checked with other seed borne fungal pathogens of wheat and rice. None of primers gave amplification with Magnaporthe grisea, a causative agent of rice blast. However, distinct RAPD profiles were obtained with Alternaria triticina, Fusarium monaliforme, Helminthosporium sativum and Rhizoctonia solani. These six arbitrary primers could distinguish T. indica, a quarantine fungal pathogen from a non-quarantine fungal pathogen, T. barclayana. The two Tilletia sps. could be discriminated not only on the basis of distinct RAPD profiles, but also by presence of few unique gene fragments amplified using all six primers.

Basidiomycota↗

PubMed2005

[Genetic polymorphism of Malassezia spp. yeast isolates from individuals with and without dermatological lesions].

INTRODUCTION: Malassezia spp. yeasts are opportunistic and newly emergent diseases. One or more species have been isolated in association with dermatological pathology and systemic disease. Their pathological role has not been fully elucidated since Malassezia spp. are common in normal skin flora. OBJECTIVE: In the current study, a search was undertaken for genetic markers in the Malassezia spp. isolates that correlate with each type of dermatologic lesion. MATERIALS AND METHODS: A total of 103 strains of Malassezia spp were isolated from patients with pytiriasis versicolor, seborrheic dermatitis, seborrheic dermatitis HIV (+), and atopic dermatitis, as well as from healthy individuals. Eight isolates from CBS Centraalbureau voor Schimmelcultures (Netherlands) were used as controls. Fingerprinting was done using random amplification of polymorphic DNA technique (RAPD) with three primers (OPA2, OPA4, OPA13). The data were analyzed with Diversity Database and SYN-TAX-PC programs. RESULTS: Intraspecies genetic heterogeneity in M. furfur, M. globosa, M. restricta, M. slooffiae, M. obtusa was observed, whereas M. sympodialis showed the greatest homogeneity. CONCLUSION: The dermatological disease caused by these different species was not associated with distinctive RAPD fingerprints.

Case-Control Studies↗

PubMed1998

Genetic typing of human and food isolates of Listeria monocytogenes from episodes of listeriosis.

Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993-1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections. The same DNA profiles within the isolates from the three episodes were obtained by both techniques. The Apal and Smal PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes. However, RAPD analysis with primer UBC-127 distinguished between these L. monocytogenes isolates.

Adult↗

PubMed2000

Molecular diversity of leuconostoc mesenteroides and leuconostoc citreum isolated from traditional french cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification.

For a long time, the identification of the Leuconostoc species has been limited by a lack of accurate biochemical and physiological tests. Here, we use a combination of RAPD, 16S rDNA sequencing, and 16S rDNA fragment amplification with specific primers to classify different leuconostocs at the species and strain level. We analysed the molecular diversity of a collection of 221 strains mainly isolated from traditional French cheeses. The majority of the strains were classified as Leuconostoc mesenteroides (83.7%) or Leuconostoc citreum (14%) using molecular techniques. Despite their presence in French cheeses, the role of L. citreum in traditional technologies has not been determined, probably because of the lack of strain identification criteria. Only one strain of Leuconostoc lactis and Leuconostoc fallax were identified in this collection, and no Weissella paramesenteroides strain was found. However, dextran negative variants of L. mesenteroides, phenotypically misclassified as W. paramesenteroides, were present. The molecular techniques used did not allow us to separate strains of the three L. mesenteroides subspecies (mesenteroides, dextranicum and cremoris). In accordance with previously published results, our findings suggest that these subspecies may be classified as biovars. Correlation found between phenotypes dextranicum and mesenteroides of L. mesenteroides and cheese technology characteristics suggests that certain strains may be better adapted to particular technological environments.

Cheese↗

PubMed2000

[Studies of the genetic polymorphism of Schistosoma japonicum (Chinese mainland strains)].

In order to analyze the interspecies diversity and the extent of diversity among Schistosoma japonicum Chinese mainland strains, the genetic variation on gene level among 6 isolates collected from Jiangxi, Hunan, Hubei, Anhui, Sichuan, Yunnan and a cultured isolate from a laboratory were studied using molecular biological technique. There were only two different bases at position 112 and 143 in 536 bp sequence of 28S rDNA-D2 domain between Anhui and Yunnan isolates, the homology was 99.6%. The result could be explained the reason of why the migration rates of single chain 28S rDNA-D2 domain among the above 7 isolates were the same completely in PCR-SSCP. However, comparing with the sequences of the Philippine isolate of S. japonicum, S. mansoni and S. aematobium, there were 6, 94 and 93 different bases and the homologies were 98.9, 82.5 and 82.7 per cent separately. With 8 restriction endonucleases to analyze the ITS of rDNA obtained by PCR from the 7 isolates, the results showed that only 3 minor bands were different, e.g. 5.3% of total 58 fragments. It was suggested that the ITS of rDNA among 7 isolates were highly conserved. Using randomly amplified polymorphic DNA (RAPD) to analyze the genetic diversity of the genomes of the 7 isolates, the average genetic distance (D) calculated from total 284 amplified fragments was 0.22. The maximum D was 0.30 and existed between Anhui and Yunnan isolates. The minimum D was 0.13 and existed between Sichuan and Yunnan isolates. The clustering analysis of genetic distances showed that the 7 isolates could be gathered in one group. From above three results, it could be considered that the genetic diversity on gene level among S. japonicum Chinese mainland strains was very low.

Animals↗

PubMed2003

Should Trypanosoma cruzi be called "cruzi" complex? a review of the parasite diversity and the potential of selecting population after in vitro culturing and mice infection.

Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

Animals↗

PubMed2005

Utility of random amplification of polymorphic DNA assay and BOX-A PCR in molecular characterization of Streptococcus pneumoniae isolates recovered from various ophthalmic infections.

BACKGROUND AND OBJECTIVES: Studies emphasizing the genotype distribution among ophthalmic isolates of Streptococcus pneumoniae are scarce. There is no conclusive information on genetic relatedness of the ophthalmic isolates with the commensal and systemic isolates of S. pneumoniae. METHODS: To find out the relatedness between the ophthalmic, invasive and commensal isolates of S. pneumoniae, genotyping was carried out by using RAPD and BOX-A PCR assays. The serotyping was carried out by a rapid co-agglutination method using 12-pooled S. pneumoniae antisera. RESULTS: Topological relationship was identified in order of similarity, and phylogenetic tree was drawn employing sequential clustering alogarithm by UPGMA linkage analysis. The 61 isolates were distributed in 17 RAPD types, 11 BOX-A types and 21 serotypes. CONCLUSIONS: Both genotyping techniques were useful in terms of intraspecies genomic variation in S. pneumoniae. BOX-A PCR typing was found to be slightly superior to RAPD analysis in terms of reproducibility and standardization. No specific S. pneumoniae serotype or genotype was found to have special predilection to cause ophthalmic infections.

Adolescent↗

PubMed1999

Genotypes of Helicobacter pylori in Polish population.

Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients. The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied. Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique. In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found. Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy. In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases. Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively. Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes. These results suggest that H. pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society. The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common. RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains. Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.

Adolescent↗

PubMed2005

Development and characterization of microsatellite markers in Cynara cardunculus L.

Cynara cardunculus L. is a species native to the Mediterranean basin that comprises 2 crops, globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC), as well as wild cardoon (var. sylvestris (Lamk) Fiori). Globe artichoke represents an important component of the South European agricultural economy but is also cultivated in North Africa, the Near East, South America, the United States, and China. Breeding activities and molecular marker studies have been, to date, extremely limited. Better knowledge of the genome of the species might be gained by developing a range of molecular markers. Here, we report on the development of 14 microsatellites (simple sequence repeats (SSRs)) through a novel approach that we have defined as the microsatellite amplified library (MAL). The approach represents a combination of amplified fragment length polymorphism and a primer extension based enriched library, is rapid, and requires no hybridization enrichment steps. The technique provided a approximately 40-fold increase in the efficiency of SSR identification compared with conventional library procedures. The developed SSRs were applied for genotyping 36 accessions of C. cardunculus, including a core of 27 varietal types of globe artichoke, 3 accessions of cultivated cardoon, and 6 Sicilian accessions of wild cardoon. Principal coordinates analysis made it possible to differentiate both cultivated and wild forms from each other.

Cynara↗