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Results for “Random Amplified Polymorphic DNA Technique”

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PubMed1997

[Emergence of Enterobacter aerogenes strains producing derepressed cephalosporinase at CHU in Limoges: molecular epidemiology by the RAPD technique].

From the beginning of the year 1996, an increase of multiresistant Enterobacter aerogenes isolates was observed. These strains overproduced their chromosomally cephalosporinase. From February to September 1996, we studied the genotypic diversity of multiresistant E. aerogenes isolates. Fifty-seven strains were analysed with the RAPD technique. These strains were isolated from 38 patients in 25 different units. These isolates were collected from urines (37), respiratory tracts (14) and wounds (6). We have observed two distinct profiles. The predominant profile (A) was recovered from 55 strains (36 patients) and the second profile (B) from 2 strains (2 patients). The clonal relatedness of strains isolated (profile A) showed an epidemic spread in our hospital. The RAPD technique is easy to perform and gives results in less than 48 h with a good discriminatory power.

Cephalosporinase↗

PubMed1999

Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers.

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16(0.35)), 0.6 (OPH-19(0.6)), and 0.65 (OPH-20(0.65)) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguished T. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium for Trichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.

Base Sequence↗

PubMed2001

Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex.

Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions that were obtained with primer OPA1, OPA1-800, OPA1- 900, and OPA1-1200, are analyzed in this study. The study revealed that each of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subsequently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, corresponding to the 3' half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact, the OPA1-800 fragment proved to be a useful DNA marker either as a DNA probe or as a target for PCR-based assays. This tool can therefore be recommended for the control of Old World Leishmania parasites, such as species discrimination, molecular tracking of isolates, or study of polymorphisms within the L. donovani species complex. Moreover, the molecular bases underlying the amplification of the RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified at least misleading observations of DNA bands amplified in a species-specific manner, in spite of their presence in the genome of the other taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions.

Animals↗

PubMed2004

Molecular epidemiology of group B streptococcal infections.

Streptococcus agalactiae (GBS) is a causative agent of sepsis and meningitis in newborns and diseases in pregnant women and nonpregnant adults. Various approaches, including both nongenetic and genetic techniques, are currently used for the study of epidemiology of GBS infections. In the present paper the different methods of molecular epidemiology of GBS infections are reviewed, and several novel approaches are introduced. The advantages and disadvantages of molecular methods are discussed and compared with traditional serotyping technique. The possible use of the molecular approaches for identification of different genetic lineages in GBS as well as for identification and control of the epidemiologically actual clones is discussed.

Animals↗

PubMed2002

Efficiency of three PCR-based markers in assessing genetic variation among cowpea (Vigna unguiculata subsp. unguiculata) landraces.

The main objective of this study was to investigate the efficiency of RAPD, AFLP, and SAMPL marker systems in detecting genetic polymorphism in cowpea landraces (Vigna unguiculata subsp. unguiculata (L.) Walp.) that probably share a similar genetic pool. A second objective was to determine the level of diversity among landraces from a restricted area, to define the most appropriate strategy of on-farm conservation. Each marker system was able to discriminate among the materials analysed, but a clear distinction between all the local varieties was only obtained with AFLP and SAMPL markers. The average diversity index was quite similar for each marker system, but owing to the differences in the effective multiplex ratio values the marker index was higher for the AFLP and SAMPL systems than for the RAPD system. The AFLP and SAMPL techniques appear to be more useful than the RAPD technique in the analysis of limited genetic diversity among the cowpea landraces tested. The significant correlations of SAMPL similarity and cophenetic matrices with those of the other markers, and the lower number of primer combinations required, indicate that this technique is the most valuable. The low genetic similarity detected among landraces suggests that all the cowpea landraces should be maintained on the respective farms from which they came.

DNA, Plant↗

PubMed2001

Optimization of random amplification of polymorphic DNA analysis for molecular subtyping of Escherichia coli O157.

Random amplification of polymorphic DNA (RAPD) analysis using the polymerase chain reaction has proved to be a useful technique in the epidemiological investigation of micro-organisms but may suffer from a lack of reproducibility in poorly optimized protocols. In this study a method of obtaining reproducible genomic fingerprints using RAPD analysis of Escherichia coli O157 is described. By systematic optimization of reaction conditions and selection of suitable primers, reproducible and discriminatory profiles could be obtained from all E. coli O157 strains tested. In addition, two other methods of obtaining reproducible profiles from E. coli O157 strains without the need to purify genomic DNA are described.

Buffers↗

PubMed2003

An effective method for axillary bud culture and RAPD analysis of cloned plants in tetraploid black locust.

An efficient micropropagation technique by axillary bud multiplication was established for cloning tetraploid black locust tree (Robinia pseudoacacia L.). The result showed that the optimal medium for shoot multiplication and elongation was Murashige and Skoog (MS) medium supplemented with 0.5 mg/l 6-benzylaminopurine in combination with 0.5 mg/l kinetin and 0.1 mg/l 1-naphthaleneacetic acid. The best medium for rooting was half-strength MS medium with 0.25 mg/l indole-3-butyric acid. In the present report, we examined the genetic fidelity of the micropropagated plants by the randomly amplified polymorphic DNA (RAPD) method with 25 primers. The cloned plants of tetraploid black locust showed complete stability.

Animal Feed↗

PubMed2005

Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversity assessment of the rice blast pathogen (Magnaporthe grisea).

This present study is the first report of the application of the retrotransposon-microsatellite amplified polymorphism (REMAP) technique in fungi. Genome fingerprinting has a major role in the characterization of population structure and in the analysis of the variability in fungi. Retrotransposon-microsatellite amplified polymorphism assay was used in virulent isolates of a rice blast pathogen (Magnaporthe grisea) as a new assay system for genetic variability studies that overcomes the limitations of previous techniques. The high polymorphism observed in REMAP could be due to past or recent actions of retrotransposon in M. grisea. Retrotransposon-microsatellite amplified polymorphism, with its superior marker utility, was concluded to be the marker of choice for characterizing M. grisea isolates.

Genetic Markers↗

PubMed2004

Phytate degradation by lactic acid bacteria and yeasts during the wholemeal dough fermentation: a 31P NMR study.

myo-Inositol hexaphosphate (IP6) is the main source of phosphorus in cereal grains, and therefore, in bakery products. Different microorganisms such as yeasts and lactic acid bacteria have phytase enzymes able to hydrolyze IP6 during the wholemeal breadmaking. In this paper, the phytase activity of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus curvatus, and Saccharomyces cerevisiae strains, isolated from southern Italian sourdoughs, is assayed using the (31)P NMR technique. The sourdough technology based on the use of lactic acid bacteria in the breadmaking is finally suggested.

6-Phytase↗

PubMed1998

Characterization of bifidobacteria by random DNA amplification.

RAPD conditions were optimized to generate reproducible banding patterns by testing primers, thermocyclers and overall reproducibility in repeat DNA analysis and separate DNA extractions. Five primers were chosen on the basis of band intensity and distribution (between 2 and 10 bands) which clearly distinguished among strains of Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis and B. longum. The use of five single-primer reactions under optimized conditions improved the resolution and accuracy of the RAPD method for the characterization of dairy-related bifidobacteria. The results indicated that this method was highly reproducible in repeated analysis. Similarity between bifidobacteria strains was evaluated based on their RAPD profiles. Using a set of five primers, it was demonstrated that it may be possible to distinguish three different species of Bifidobacterium (B. breve, B. bifidum and B. adolescentis), based on similarity of the RAPD profiles to known reference strains. Furthermore, application of the RAPD technique may also be useful and faster, than traditional systematics for placement of industrial strains into specific clusters (either B. longum/infantis or B. animalis/lactis).

Animals↗

PubMed2003

Genetic variation in Oryza species detected by MITE-AFLP.

MITE-AFLP markers were successfully used to study the genetic variation and species relationship in Oryza species. Analysis of 53 accessions of Oryza species with seven MITE-AFLP primer combinations detected a total of 250 polymorphic fragments. High polymorphism was detected within and between Oryza species. Species relationships were analyzed by the pattern of presence or absence of homologous fragments, because nucleotide sequences of the detected MITE-AFLP fragments revealed that the same fragments in different species shared very high sequence homology. The genetic distances (GDs) between species were higher than those within species and the GDs in O. sativa complex were higher than those in O. officinalis complex. The phylogenetic tree recognized two major groups at 62% genetic similarity; group I consists of all AA genome species of the O. sativa complex, and group II consists of BB-, CC-, EE- and BBCC genome species of the O. officinalis complex. Therefore, this study demonstrated that the MITE-AFLP technique provide a tool for studying the genetic variation and species relationship in Oryza species.

DNA Transposable Elements↗

PubMed2003

Mutans streptococci strains prevalence before and after cavity preparation during Atraumatic Restorative Treatment.

Critics argue that all carious dentine is not removed from the hand-prepared cavity during the Atraumatic Restorative Treatment (ART) procedure, and that the caries process is soon resumed. The aim of this study was to determine the effectiveness of ART in removing carious tissue, by investigating the numbers of mutans streptococci and lactobacilli, with emphasis on the prevalence of Streptococcus mutans and Streptococcus sobrinus strains before, and after ART treatment of dental caries. Two microbiology samples were collected. The first sample was removed from the centre of the carious lesion at the enamel-dentine junction, and the second was collected from the centre of the hard cavity wall above the pulp, after the soft infected dentine had been manually removed. A total of 71 mutans streptococci isolates from 31 children and 40 carious teeth were subcultured, biochemically characterised and genotyped by the arbitrarily primed polymerase chain reaction (AP-PCR). Results showed a significant decrease in TVC (P<0.0001), mutans streptococci (P < 0.0001) and lactobacilli (P = 0.0002) after cavity preparation. AP-PCR identified S. mutans strains that were undetectable during biotyping, and divided clinical isolates into two main clusters. In all, 63% (45/71) of isolates from the carious lesions comprised S. mutans strains. After cavity preparation, this was reduced to 35% (25/71), of which 30% (21/71) were S. mutans and the remaining 6% (4/71) S. sobrinus strains. The number of mutans streptococci strains was below detectable levels in 19 of the prepared cavities. The significant decrease in bacteria after manual cavity preparation demonstrates the reliability of a standardized ART technique, yet the presence of S. mutans strains shows that the effectiveness of the ART procedure can vary during treatment and between dental practitioners.

Child↗

PubMed1994

Differentiation of poplar and willow clones using RAPD fingerprints.

The technique of random amplified polymorphic DNA (RAPD) fingerprinting was used to differentiate species and identify clones of 15 poplar and 15 willow clones (ramets of three poplar clones were also included to verify the stability of the results). Four random DNA primers (Deca-11, Chl-1, Chl-4 and Chl-10) and an M13 universal primer were used to determine DNA polymorphism among the clones. Based on the DNA banding pattern obtained with the four DNA primers by polymerase chain reaction (PCR) amplification we conclude that RAPD fingerprinting with properly selected primers can be used for clonal and species differentiation of poplar and willow. The technique is simple, accurate and inexpensive.

Journal Article↗

PubMed1997

Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans.

The genetic diversity of 47 enterotoxigenic Escherichia coli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD). Informative band arrays were obtained with three 10-mer primers with G+C contents of 50, 60, and 70%. Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established. The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil. Moreover, the RAPD technique revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed. These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.

Brazil↗

PubMed2003

Epidemiology of Streptococcus suis serotype 5 infection in a pig herd with and without clinical disease.

The aim of this study was to describe the transmission and the kinetics of the infection caused by Streptococcus suis serotype 5 in a multisite farrow-to-finish pig herd. Most sows carried S. suis serotype 5 in their vaginal tract, but not in their nasal cavities, as demonstrated by immunomagnetic separation (IMS) technique. Their offspring became infected during farrowing, confirming vertical transmission. During the first 4 weeks of life, a low number of piglets were carriers of S. suis serotype 5 in their nasal cavities. However, when clinical signs appeared, the carrier rate significantly increased, suggesting that isolation from nasal cavities is a better indication of active transmission than of a carrier state. Clinical cases were present in animals between 4 and 8 weeks of age, when maternal antibodies were at their lowest level. Up to six different genotypes of the same serotype could be identified by random amplified polymorphic DNA; however, a single clone was responsible for all clinical cases studied. This clone could only be isolated from a single sow, indicating that its prevalence in breeding animals was low. Interestingly, 1 year later, clinical disease associated with S. suis serotype 5 spontaneously disappeared. At that time, the genotype responsible for the clinical signs was not detected in the herd and the levels of antibodies in sows and maternal antibodies in piglets were not higher than those of the previous year.

Animals↗

PubMed1995

Detection of hepatitis B virus transcriptional activity: an hypothesis.

This paper outlines a method of demonstrating hepatitis B virus (HBV)-related transcriptional activity based on the random amplification of cDNA ends (RACE) protocol. Following RNA extraction from the appropriate tissue, cDNA is synthesized using a long oligo d(T) based primer containing unique 5' oligonucleotide sequences. Nested polymerase chain reaction (PCR) is then carried out using virus-specific oligonucleotides and standard PCR (approximately 20 base pairs) oligonucleotides homologous to 5' regions of the long oligo-d(T) primer used to initiate the cDNA reaction. This technique is particularly applicable to the study of HBV transcription in small samples of tissue, for example, from renal biopsies obtained from patients with HBV-related nephritis.

Base Composition↗

PubMed2001

[Genetic analysis of mutant genes and RAPD marker of mutant gene of narrow leaflet in soybean].

Mutant E182 with "narrow leaflet-4 seeded pod" was selected from descendents of EMS-treated seeds of soybean variety Lu Dou No. 4 (LD4) with "ovate leaflet-without 4 seeded pod". Genetic analyses of F2 individuals of crossing between mutant E182 and parent LD4 indicated that segregation ratio between ovate and narrow leaflet was 3:1, so was segregation ratio between "without 4 seeded pod" and "4 seeded pod". Segregation ratios of four character types in F2 population of 1,654 individuals were beyond 9:3:3:1 of two pairs of independent gene, showing linkage inheritance. Reccmbinant ratio between mutant genes of narrow leaflet and 4 seeded pod was 11.24% +/- 0.81%. On the other hand, mutant E182, parent LD4 and F2, F3 individuals of their hybrids were analyzed by means of RAPD technique. The marker OPY6-1300 linked with the mutant gene of narrow leaflet was generated, and genetic distance of the marker and mutant gene was 8 cent Morgan (cM), being 10 cM nearer than RFLP marker of narrow leaflet generated by shoemaker.

Genetic Markers↗

PubMed2000

Delimination of brewing yeast strains using different molecular techniques.

In general, the genetic characteristics, the phenotype and the microbial purity of the production brewing yeast strains are among the most important factors in maintaining a consistently good quality of products. Analysis of restriction fragment length polymorphism (RFLP) patterns of 18S rRNA-coding DNA was investigated to group ale and lager strains. All production brewing yeast strains showed the same RFLP pattern as the type strain and synonym type strains of S. cerevisiae, and were quite different from the type and synonym type strains of S. pastorianus. Based on these data, all production brewing yeast strains investigated in this study appeared to belong to S. cerevisiae. Electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis appeared to be suitable methods for distinguishing not only the type and synonym type strain of S. cerevisiae and S. pastorianus, but also the ale and the lager strains.

Beer↗