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PubMed1995

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila.

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida, and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.

Aeromonas↗
PubMed2000

Complement-resistant Moraxella catarrhalis forms a genetically distinct lineage within the species.

Moraxella catarrhalis is a bacterial species that has been implicated in 15-20% of all cases of otitis media in the USA and the complement-resistant variant of M. catarrhalis has been considered particularly pathogenic. A collection of geographically diverse, complement-sensitive (n=28) and -resistant strains (n=47) of M. catarrhalis was assembled in order to analyse the bacterial population structure. All strains were identified as M. catarrhalis by conventional microbiological and biochemical methods. Amplification of the small subunit (ssu) ribosomal RNA gene followed by restriction fragment length polymorphism (RFLP) analysis did not reveal consistent differences between serum-susceptible and -resistant M. catarrhalis isolates. Interestingly, upon automated ribotyping using the Qualicon RiboPrinter(R) microbial characterisation system, the complement-sensitive and -resistant strains segregated into two groups. This suggested the existence of two clearly distinguishable lineages within the species M. catarrhalis. This observation was corroborated by pulsed field gel electrophoresis (PFGE) of DNA macro-restriction fragments, a non-ribosomal PCR RFLP procedure and random amplification of polymorphic DNA (RAPD) analysis. All procedures grouped the two variants similarly. Redefinition of the taxonomic status of complement-resistant M. catarrhalis or even the definition of a new species may be opportune.

Bacterial Typing Techniques↗
PubMed2003

Comparison of traditional and molecular typing methods of Escherichia coli O157.

An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.

Bacterial Typing Techniques↗
PubMed1993

PCR with arbitrary primers: approach with care.

New techniques have recently been described that employ the polymerase chain reaction (PCR) to amplify arbitrary regions of a genome using a single primer. The techniques reveal polymorphisms in insect taxa that lack allozyme variation and, for the first time, permit genetic polymorphisms to be rapidly analysed in small arthropods (e.g. mites, endoparasitic wasps). The methods have been used in identification of sub-species and cryptic species, and have applications in population genetics and genetic fingerprinting. They are fairly inexpensive, do not require the use of radioactivity, are relatively simple to learn and can easily be adapted to most laboratories. However, their application is not without technical problems and practical limitations. The purpose of this note is to indicate the critical factors to consider before launching into their use. We chiefly emphasize that most polymorphisms revealed by these methods segregate as dominant markers. Furthermore, application of these techniques requires extensive standardization and may not prove to be reproducible among various laboratories especially those employing different types of thermal cyclers. There are some unique features of these polymorphisms to consider when using them in genetic fingerprinting. In addition, because the techniques amplify arbitrary regions of genomes, similarly sized fragments amplified between two species may not be homologous. This argument and empirical observations suggest that PCR with arbitrary primers will have limited application in molecular systematics above the intraspecific level.

Animals↗
PubMed2001

Variation in random amplified polymorphic DNA (RAPD) profiles specific to fluconazole-resistant and -sensitive strains of Candida albicans.

Random amplified polymorphic DNA analysis was used to detect genotype relatedness among clinical fluconazole-resistant and -sensitive strains of Candida albicans recovered from twenty HIV-infected patients having oropharyngeal candidiasis. Sensitive strains were obtained from a local hospital and were from patients that had not been treated with azole drugs while resistant strains were recovered from patients in different parts of Europe and their resistance was a consequence of drug-treatment given to the patients. On amplification with different arbitrary sequence decamer primers, the results demonstrated a homogeneous banding pattern for all sensitive strains that was distinct from that obtained in case of the resistant strains. The DNA profiles of strains were thus broadly clustered into two major groups of resistant and sensitive strains. The RAPD technique may be useful in differentiating fluconazole-resistant strains from the -sensitive ones for early identification of resistant isolates from AIDS patients.

AIDS-Related Opportunistic Infections↗
PubMed2001

Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa.

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.

Bacterial Typing Techniques↗
PubMed1997

DNA profiling resolves the limited importance of flower colour in defining varieties of Lantana camara.

Randomly amplified polymorphic DNA (RAPD), successfully used to establish flower colour, is of limited importance in characterising weedy varieties of Lantana camara. Initially the internal transcribed spacer 1 (ITS1) region was sequenced for common pink and common pink-edged red varieties of L. camara from Australia and L. urticofolia from the neotropics. This proved unhelpful in differentiating varieties due to a lack of variation and the hybrid origin of L. camara, necessitating the utilisation of DNA profiling techniques. Unweighted pair group method arithmetic average (UPGMA) analysis of RAPD data demonstrated that geographical proximity contributes more significantly to genetic relatedness than flower colour. Analysis of molecular variance (AMOVA) likewise demonstrated that geography accounts for a relatively large variance component. These data indicate that the use of flower colour as a primary identification tool needs to be reevaluated. The use of RAPD may prove useful in characterising the weedy varieties of lantana present in Australia and the South Pacific. Since biological control efforts are being hindered by the inadequacy of current morphological taxonomy, it is expected that DNA profiling will underpin continuing studies on the management and control of L. camara.

Australia↗
PubMed2003

DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources.

AIMS: To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2. METHODS AND RESULTS: Eleven human and food strains isolated over a 15-year period were examined. All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant. Ten strains had VT2 genes which were all vtx2d. Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory. Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. CONCLUSIONS: As a result of apparent clonality, PFGE proved essential for differentiation. Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.

Adhesins, Bacterial↗
PubMed2004

The use of RAPD in ecotoxicology.

Toxic compounds may interfere with the genetic constitution of populations, either directly through mutagenic activity, or indirectly via population-mediated processes (i.e. selection, bottleneck). These processes are initiated when toxic compounds reduce the survival and/or fecundity of exposed organisms, either through the accumulation of unfavorable mutations or when they adversely affect the physiology of an organism and/or the environment in which it has to survive. In this review, we describe how the RAPD technique can be applied in an ecotoxicological context, providing information on all direct and indirect routes through which toxicants may affect the genetic structure of populations. Based on RAPD band intensity, gain/loss and band numbers, three major types of RAPD fingerprint analyses are discussed, yielding diagnostic, phenetic and genetic information. Ecotoxicological literature examples demonstrate that, under strictly standardized conditions, the RAPD technique can be a useful tool to preliminary assess toxicological population genetic effects, particularly since this technique is relatively inexpensive and yields information on a large number of loci without having to obtain sequence data for primer design. However, currently only a small fraction of its potential is used in ecotoxicology. Statistical tools and parameters, as used in other RAPD studies, should be applied in ecotoxicological research as well in order to fully exploit the potential of this technique. Finally, due to their random nature, RAPD data often must be considered as preliminary until they are further documented by cloning, sequencing and probing techniques.

Animals↗
PubMed2004

[Research advance in application of RAPD techniques in entomology].

This paper summarized the application of RAPD techniques in insect taxology and ecology, relative of species, systemic development, identification of pest, diagnosing for pest resistance, construction of molecular linkage map, and molecular assistant breeding. The problems existing in the applications of RAPD in entomology were indicated, and their countermeasures were given. It was mentioned that with the development of theories and experimental techniques, insect molecular biology would make new progress and conquer all those problems.

Animals↗
PubMed2003

Female-specific DNA sequences in geese.

1. The OPAE random primers (Operon Technologies, Inc., CA) were used for random amplified polymorphic DNA (RAPD) fingerprinting in Chinese, White Roman and Landaise geese. One of these primers, OPAE-06, produced a 938-bp sex-specific fragment in all females and in no males of Chinese geese only. 2. A novel female-specific DNA sequence in Chinese goose was cloned and sequenced. Two primers, CGSex-F and CGSex-R, were designed in order to amplify a 912-bp sex-specific polymerase chain reaction (PCR) fragment on genomic DNA from female geese. 3. It was shown that a simple and effective PCR-based sexing technique could be used in the three goose breeds studied. 4. Nucleotide sequencing of the sex-specific fragments in White Roman and Landaise geese was performed and sequence differences were observed among these three breeds.

Animals↗
PubMed2002

Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced.

Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab. They were biochemically identified using API20NE and confirmed by a PCR-based assay. The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV. All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis. Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure. The polysaccharides were analysed by means of methylation analysis and 1H-NMR spectroscopy in order to determine structural similarities. It was shown that different strains are capable of producing chemically different polysaccharides.

Adolescent↗
PubMed2002

Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comté cheese ripening.

This study investigated the genotypic characteristics of selected and wild homofermentative thermophilic lactobacilli strains during ripening of Comté cheeses, made into two cheese plants. Both amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) and classical biochemical tests were used to identify isolates. Diversity within homofermentative lactobacilli was not found in their species composition since the same two species Lactobacillus helveticus and L. delbrueckii susbp. lactis were isolated from cheeses. In cheeses made with natural whey starter, it appeared that the most likely sources of L. helveticus and L. delbrueckii susbp. lactis were the starter and raw milk, respectively. The examination of RAPD profiles of lactobacilli strains revealed 19 RAPD groups among 50 isolates, which were different from selected starter strains. Using RAPD, REP-PCR, and PFGE to identify selected starter strains during cheese ripening, we showed that L. helveticus decreased quickly while L. delbrueckii susbp. lactis sustained high viability during ripening. The use of selected L. delbrueckii susbp. lactis strains diminished the genetic diversity among strains isolated from cheese, probably in preventing the raw milk microflora from growing.

Bacterial Typing Techniques↗
PubMed2001

PCR as a diagnostic and quantitative technique in veterinary parasitology.

Over the past 15 years, there has been a dramatic evolution in molecular approaches to study parasites and parasitic diseases. Many of these advancements have been brought about through the development of new applications of the polymerase chain reaction (PCR). Enhancements in sensitivity that can be achieved using PCR now permit scientists to investigate changes at the level of a single cell, far below what is often needed for parasite-derived applications. PCR has had a substantial impact on advances made in the areas of parasite systematics and epidemiology, immunology and host-parasite interactions, recombinant DNA vaccine development and most recently, the analysis of whole genomes either through directly sequencing the DNA, the analysis of expressed sequence tags (ESTs) or through the rapidly growing field of functional genomics. This paper, however, focuses on the application of PCR methodology to parasite detection and differentiation, and the diagnosis of disease. Specific attention is given to advances provided by multiplex PCR, fluorescence-based "real-time" PCR, and the utilization of PCR as a quantitative technique.

Animals↗
PubMed1995

Typing of Listeria monocytogenes by random amplified polymorphic DNA (RAPD) analysis.

The aim of the study was to determine the effectiveness of random amplified polymorphic DNA analysis in typing Listeria monocytogenes from human infections. Twenty-five L. monocytogenes serogroup 1/2 and 70 serogroup 4 including 14 serovar 4b(x) were typed by RAPD-PCR analysis. Six primers were used to type each L. monocytogenes isolate and the DNA amplification performed with supertaq DNA polymerase in a Hybaid Thermal Reactor. Each bacterial strain was analysed separately with all primers and the profiles were judged by eye and designated to a group by comparison to other strains. Bands were classified as major or minor. Based on analysis of major band patterns, the 25 serogroup 1/2 isolates gave rise to 12 different groups. The groups only contained serovar 1/2a or 1/2b with a single exception. Using minor bands all isolates could be distinguished. All the serogroup 4 isolates gave the same major band patterns. The 14 serovar 4b(x) isolates which were epidemiologically related gave identical profiles with the exception of one isolate. Of the remaining strains, 41 produced individual patterns on minor band analysis. RAPD analysis with multiple primers is low cost, discriminatory and is most ideally suited to testing small (< 50) numbers of strains. We have shown that serogroup 1/2 L. monocytogenes strains are a more diverse group than serovar 4b strains and RAPD-PCR will provide a technique of considerable value in typing L. monocytogenes in the future.

Bacteriophage Typing↗