RAPD analysis, serotyping, and esterase typing indicate that the population of Listeria monocytogenes strains recovered from cheese and from patients with listeriosis in Belgium are different.

Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13). High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960). The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing. RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases. Our analysis indicates that the population of strains of L. monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period.     

Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences

Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.     

Potential of Three-Way Randomly Amplified Polymorphic DNA Analysis as a Typing Method for Twelve Salmonella Serotypes

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA

A method was developed to obtain reproducible DNA fingerprints from Campylobacter by PCR-based amplification, without the need to isolate total DNA. Randomly amplified polymorphic DNA (RAPD) profiles were generated with three randomly designed 10-mers, using each separately as an amplification primer. A range of C. jejuni serotypes could be typed by RAPD analysis. Depending on the primer, the analysis of RAPD profiles resulted in different levels of discrimination between the strains. Clear correlations were observed between results of RAPD analysis and serotyping. Two of the primers tested generated RAPD profiles which allowed discrimination of strains within given Penner and Lior serotypes.     

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.     

Rapid differentiation between Octopus vulgaris Cuvier (1797) and Octopus mimus Gould (1852), using randomly amplified polymorphic DNA

Seventeen specimens of Octopus vulgaris and Octopus mimus were investigated using randomly amplified polymorphic DNA (RAPD). Clearly differentiable RAPD fingerprints allowed a fast and reliable genotypic discrimination of these species. Thus, molecular genetic evidence from total DNA complements recent results of a comparative analysis of mitochondrial sequence data supporting the taxonomic separation of Octopus mimus and Octopus vulgaris .     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.     

Utility of a pre-optimized kit for random amplified polymorphic DNA in typing Candida albicans

The utility of a pre-optimized kit for random amplified polymorphic DNA (RAPD) was assessed in typing diverse strains of Candida albicans from epidemiologically unrelated inpatients (interpatient analysis) and in detecting clonal variations that maybe present within individual patient isolates (intrapatient analysis). Stool samples from inpatients were cultured on Inhibitory Mold agar. Nine individual colonies from all patients with > or =9 colonies of C. albicans (n = 18) were selected, frozen, and karyotyped using CHEF genomic DNA plug kits and CHEF-DRIII. Each of the selected colonies was then analyzed by RAPD, utilizing the selected kit, with 6 primers. Interpatient analysis revealed 9 karyotypes and 17 RAPD composites. RAPD discrimination was significantly better (p < 0.001). Intrapatient analysis revealed 34 (21%) and 33 (20.4%) variants among 162 colonies tested by RAPD and karyotyping, respectively. The results were discordant in 25 variants, all with differences of 1-3 bands. These results illustrate that this pre-optimized kit for RAPD provides excellent discrimination of genetically unrelated strains. Its performance in delineating subtle clonal differences was comparable with karyotyping; both methods failed to detect all minor genetic variations. The ease of use and quick turnaround time of this kit offer a practical and reliable method for typing diverse strains of C. albicans, but may be inadequate for assessing microevolution.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes

CH. NIEDERHAUSER, CH. HÖFELEIN, M. ALLMANN, P. BURKHALTER, J. LÜTHY AND U. CANDRIAN. 1994. The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.     

Sub-typing of animal and human Campylobacter spp. using RAPD

R.H. MADDEN, L. MORAN AND P. SCATES. 1996. Based on a 10-mer primer (5'- CCTGTTAGCC-3'), a random amplified polymorphic DNA (RAPD) method for typing Campylobacter coli isolated from pigs was developed. The method proved effective with a high discrimination and good reproducibility. In contrast with serotyping no untypable strains were found out of a total of 269 isolates (veterinary, food and clinical) examined. The method was also successfully applied to typing Campylobacter jejuni from a similar range of sources.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

A comparison of molecular typing methods for Moraxella catarrhalis

AIMS: Three molecular typing techniques were examined to determine which method was the most discriminatory in order to perform epidemiological typing of Moraxella catarrhalis. METHODS AND RESULTS: Twenty-five Mor. catarrhalis isolates obtained from nasopharyngeal aspirates collected from Aboriginal and non-Aboriginal children were subjected to random amplified polymorphic DNA (RAPD) analysis, automated ribotyping and pulsed field gel electrophoresis (PFGE). RAPD analysis determined two Mor. catarrhalis types, automated ribotyping with PstI determined four Mor. catarrhalis ribogroups and PFGE analysis with NotI determined 21 pulse field groups within the 25 isolates examined. CONCLUSIONS: Analysis of discrimination index and typeability demonstrated that PFGE is the most discriminatory method for typing Mor. catarrhalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that PFGE is the most appropriate molecular tool for the epidemiological study of Mor. catarrhalis.     

Evaluation of three methods for DNA fingerprinting of Corynebacterium pseudotuberculosis strains isolated from goats in Poland

Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.