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Evaluation of two PCR-based techniques for molecular epidemiology in Finland, a high-endemic area with four sympatric Trichinella species.

Trichinella larvae collected from wildlife, domestic and synanthropic animals in Finland were identified to species by two molecular techniques: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and the recently described multiplex PCR. The RAPD-PCR was very sensitive to the sub-optimal preservation muscle larvae and resulting in weak and smeared bands on the gels for such material. However, the same samples yielded easily recognizable bands in the multiplex PCR; this latter technique is then recommended for epidemiological studies, especially when the preservation of the samples is sub-optimal. For larvae in good condition the unequivocal bands obtained by multiplex was the easiest identifiable. Four species of Trichinella were identified in the material: T. spiralis, T. nativa, T. britovi, and T. pseudospiralis. Trichinella britovi is a new record for Finland, and T. pseudospiralis is a new record for Northern Europe. Mixed infections between T. britovi and T. spiralis, T. nativa and T. spiralis, and between T. britovi and T. nativa were detected; this is the first record of a mixed infection between T. spiralis and T. nativa in a naturally infected host. Raccoon dogs were the only host species from which all of the four Trichinella species were detected. Trichinella spiralis was found in both domestic animals and wildlife, but none of the sylvatic Trichinella species were detected in domestic pig.

Animals↗

Differentiation of Lycium barbarum from its related Lycium species using random amplified polymorphic DNA.

The RAPD (random amplified polymorphic DNA) technique was applied for the first time to distinguish Lycium barbarum from other closely related species of the same genus. In this study, eight samples were collected, including five species, two varieties and one cultivated variety. A total of fifty arbitrary primers were used in the RAPD analysis. Distinctive DNA fingerprints corresponding to different Lycium species were successfully obtained from ten primers. Similarity index (S.I.) analysis revealed that the values are higher between intraspecies than interspecies. These results confirmed that the RAPD technique can be employed for distinguishing closely related species of Lycium.

Base Sequence↗

Genetic characterization of the three medicinal Echinacea species using RAPD analysis.

The three medicinal species of the Echinacea genus, E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench were distinguished using the RAPD (random amplified polymorphic DNA) technique. Species-specific markers were identified from amplicons obtained with four of the twenty 10-mer primers contained in the Operon RAPD kit A. In particular, one marker was identified for E. angustifolia (OPA 20, 1800 pb) and E. pallida (OPA 10, 600 pb) and three markers for E. purpurea (OPA 11 : 1250 pb; OPA 17 : 750, 1800 pb). Genetic distance analysis indicated a high degree of difference among the three species with a relative lower difference between E. angustifolia and E. pallida.

Echinacea↗

["Arbitrarily primed PCR". Or: chance in the service of science].

"Arbitrarily primed PCR" (AP-PCR) methods are based on the amplification of DNA with arbitrarily selected primers. In the present review article two selected applications of this methodical approach are summarized. Using the RAPD ("Random Amplified Polymorphic DNA") technique the genetic variability of different organisms can be analyzed by generating a genomic fingerprint. Echinococcus granulosus isolates (metacestodes) from Spain and Switzerland were comparatively characterized by the use of this molecular epidemiological tool. The following groups of genetically related isolates could be identified: (a) equine isolates (horse/donkey) from Spain and Switzerland, (b) cattle isolates from Switzerland, (c) goat isolates from Spain and pig isolates from both countries, (d) sheep, cattle and human isolates from Spain. Isolates derived from Swiss patients were forming a separate group with respect to their genetic relatedness. As a further application of the AP-PCR, the development of a novel technique for the construction of cDNA libraries from minute amounts of starting material using an Echinococcus multilocularis metacestode library as an example is presented.

Animals↗

Molecular characterization of invasive and noninvasive Campylobacter jejuni and Campylobacter coli isolates.

Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of Campylobacter diarrhea. Molecular characterization of invasive and noninvasive Campylobacter isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%) Campylobacter isolates (P = 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the iam locus was amplified in 85% of invasive and 20% of noninvasive strains (P = 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire iam locus showed a HindIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive Campylobacter strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the iam locus.

Bacterial Proteins↗

Determination of the components in a Chinese prescription, yu-ping-feng san, by RAPD analysis.

In this study, the RAPD (random amplified polymorphic DNA) technique was employed for the first time to determine the components in a Chinese herbal prescription. Forty decamer oligonucleotide primers were screened in the RAPD analysis to identify three Chinese medicines, the dried root of Astragalus membranaceus (Fisch.) Bge., the dried root of Ledebouriella seseloides Wolff, and the dried rhizome of Atractylodes macrocephala Koidz, in a Chinese prescription. Only primer OPP-10 simultaneously generated three distinct markers were each specific to one component. The marker with 200 bp is specific to Astragalus membranaceus; the 440 bp marker is specific to Atractylodes macrocephala; and the remaining marker with 500 bp was present in Ledebouriella seseloides. The presence of the three herbal medicines in the mixed sample, the Chinese prescription, was determined when the primer OPP-10 RAPD reaction was performed. The technique was proved to contribute to the identification of components in the Chinese medicinal preparations.

Astragalus propinquus↗

Characterization of Listeria monocytogenes and Listeria innocua from a vegetable processing plant by RAPD and REA.

The incidence of Listeria monocytogenes in a vegetable processing plant was investigated over a 23-month period. Frozen ready-to-eat vegetable samples, well as the plant environment, were sampled. The molecular subtyping techniques, Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Analyses (REA), were performed to help investigate the origin and routes of Listeria dissemination. The low and sporadic incidence of L. monocytogenes made it impossible to establish an epidemiological sequence in the processing plant, though a case of cross-contamination between tomato and ratatouille was detected. Listeria innocua subtyping, however, allowed us to determine the prevalence of several strains in vegetables, and their presence on machinery samples suggested the possibility of cross-contamination during processing. The low incidence of L. monocytogenes indicated that the risk of listeriosis transmission by vegetable consumption is low. On the other hand, the isolation of the same strain of L. innocua in several surveys pointed out the risk of colonisation on surfaces and machinery. The persistence of Listeria spp. is a cause for concern as can lead to future contamination of vegetables processed in the plant and to a possible increased risk for health. Therefore, periodic controls for the presence of Listeria spp. and a further review of the cleaning and disinfection procedures used in frozen vegetable plants are recommended.

Bacterial Typing Techniques↗

The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species specific PCR-primers based on diagnostic RAPD bands.

The randomly amplified polymorphic DNA technique (RAPD) is a modification of PCR that uses short, arbitrarily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population- or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/Maxam) and chain termination (Sanger) techniques. Based on this sequence information, highly specific oligonucleotide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculans.

Ascomycota↗

Echinococcus granulosus: genomic and isoenzymatic study of Spanish strains isolated from different intermediate hosts.

The phenomenon on intraspecific variation in Echinococcus granulosus is already documented in Spain, where unilocular hydatidosis is an endemic disease. The first speciation studies, focused at a genomic level, showed the existence of three different strains: ovine-bovine-human, equine and swine-caprine. In the present study, the genomic identification, by random amplified polymorphic DNA technique (RAPD) of a larger number of Spanish E. granulosus isolates, using five different primers, showed the maintenance of these groups. Thus, some of these strains may not be infective for man. These conclusions were supported by a phenotypic characterization of the same isolates by zymodeme technique, showing the five isoenzyme systems used that Spanish E. granulosus strains can also be distinguished at a phenotypic level by isoenzymatic patterns. Both techniques (RAPD and zymodemes) were used for statistical analysis and for the construction of two dendrograms, which were slightly different. In addition, some intrastrain variation was detected with both techniques, a phenomenon that is directly related to the different speciation theories proposed for E. granulosus strains. The epidemiological implications of the results are discussed in the text.

Animals↗

Molecular typing of the Legionella pneumophila population isolated from several locations in a contaminated water network.

The RAPD (random amplified polymorphic DNA) technique has been developed for the molecular typing of Legionella in order to characterise the populations of hot water systems. During this study, 22 primers were tested and the four most informative ones were selected. The optimisation of the PCR conditions allowed the setting up of a powerful discriminative genotyping method. Moreover, the definition of a quality management method allowed definition of the key steps and the number of replicates to ensure reproducibility of the RAPD pattern. The RAPD was used to study the hot water network of a building. Legionella colonies (91) were isolated from seven locations and genotyped. The diversity of the population in one sample could vary from one to seven different strains. The study of the traceability showed that, in most of the cases, different populations could be present at different locations of the same network.

Genotype↗

Use of RAPD (random amplified polymorphic DNA) to analyse genetic diversity of dematiaceous fungal pathogens.

Thirteen strains of chromoblastomycosis and phaeohyphomycosis etiologic agent fungi were obtained from different geographical origins. These strains were genotypically compared by means of the RAPD (Random Amplified Polymorphic DNA) technique. The data generated showed a high degree of polymorphism between distinct species and a low polymorphism between strains of the same species. The results generated by these tests were subjected to a numerical taxonomy analysis, using the unweighted pair-group method. A phenogram was constructed for the set of strains studied. Based on its structure, we concluded that genotypical data provide enough information to us to use the unweighted pair-group method to cluster the strains in accordance to their respective species. The phenogram grouped in a single branch the strains of Fonsecaea pedrosoi and F. compacta species, indicating a great similarity between these fungi, and suggesting that the classification as distinct species may not be appropriate for these species of the genus Fonsecaea.

Chromoblastomycosis↗

Genetic diversity among clinical isolates of Candida glabrata analyzed by randomly amplified polymorphic DNA and multilocus enzyme electrophoresis analyses.

The genetic diversity of 47 clinical and reference strains of Candida glabrata from several geographical origins and diverse clinical disorders, with different antifungal susceptibilities, as well as their genetic relationships were studied through multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) techniques. The genetic diversity estimated for 11 MLEE loci measured as average heterozygosity (h) was 0.055. A high level of genetic relatedness among isolates was established by cluster analysis. Forty-nine RAPD markers were analyzed, and the average genetic diversity among isolates, estimated by Shannon's index (Ho), was 0.372. The PhiST values estimated through an analysis of molecular variance to assess genetic differentiation among isolates revealed no genetic differentiation among them. Our results revealed very low genetic diversity among isolates, a lack of differentiation, and no association with their geographic origin and the clinical characteristics.

Antifungal Agents↗

Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula.

Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.

Animals↗

Last male sperm precedence in a damselfly demonstrated by RAPD profiling.

We used the random amplified polymorphic DNA technique to determine last male sperm precedence (P2) in the damselfly Calopteryx splendens xanthostoma (Charpentier). We amplified DNA from mothers, putative fathers and from the embryos of individual offspring, and subsequently calculated band-matching coefficients between known first-order relatives (offspring within a clutch) and non-relatives (mothers and fathers) to estimate last-male paternity. The data indicate that, as in other Calopterygidae, P2 is high (0.98) in the bout of oviposition immediately following copulation, despite the fact that the males of this species do not completely remove the sperm of previous males (Siva-Jothy & Hooper 1995).

Animals↗

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA.

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.

Animals↗

Identification of Candida species by randomly amplified polymorphic DNA fingerprinting of colony lysates.

We have characterized a method that produces simple yet diagnostic fingerprints that are unique to isolates of Candida species. DNA from individual colonies can be amplified from crude single-colony lysates. Randomly amplified polymorphic DNA (RAPD) fingerprints generated from a single primer correctly identified the species of most (>98%) of the isolates identified with CHROMagar Candida plates as non-Candida albicans Candida species. RAPD fingerprints were much more informative than the plates, since they distinguished between all tested species and required less time. Most (91%) of these identifications agreed with those assigned by API 20C tests. In almost every incident of species identity mismatch, electrophoretic karyotyping showed that the RAPD fingerprint was correct. This underscores the improved objectivity and reliability of this method over those of conventional diagnostic tools. The identities of approximately 30% of C. albicans isolates identified in clinical laboratories by positive germ tube tests are not verified by either testing on CHROMagar Candida plates or RAPD fingerprinting. Data suggest that clinical isolates conventionally identified as C. albicans in clinical settings are heterogeneous, consisting of both misidentified and atypical yeasts. RAPD fingerprints obtained from primary culture plate colonies allows for rapid, highly accurate determinations of Candida species, hence permitting earlier selection of appropriate antifungal agents in the clinical setting.

Candida↗

Tracing the geographic origin of the cosmopolitan parthenogenetic insect pest Liposcelis bostrychophila (Psocoptera: Liposcelididae).

The randomly amplified polymorphic DNA technique was used to trace the geographic origin of Liposcelis bostrychophila Badonnel populations in Australia from unknown geographic sources internationally. Haplotype (or clonal) diversity was high, with 474 unique haplotypes found from 616 individuals genotyped. Gene diversity estimates (0.10-0.28) and percent polymorphic loci (38.1-88.1%) were moderate to high for most populations. This resulted in genetic distance estimates that ranged from 0.04 to 0.26 and were significantly different for most pairwise population combinations. G ST values for all populations were also moderate (0.04-0.54) and again were significantly different for most pairwise population comparisons. Analysis of molecular variance revealed that the majority of variation was apportioned among individuals within populations regardless of the level at which they were grouped. Gene flow (Nm) was mostly low for all pairwise populations comparisons with an average Nm=1.8. A non-significant negative correlation between genetic distance and geographic distance was found for worldwide populations. In contrast, within Australian populations a significant positive correlation between genetic distance and geographic distance was detected. Genetic relationships explored using unweighted pair group method analysis and non-metric multidimensional scaling indicated a mixed pattern of genetic similarities among all populations. Multiple introductions, from a wide range of international source populations, have obscured the ability to accurately determine the geographic origin of L. bostrychophila in Australia.

Animals↗

Molecular analysis of variability within the toxigenic Aspergillus ochraceus species.

Genetic variability of Aspergillus ochraceus was examined at the DNA level. Based on the HaeIII-Bg/II generated mitochondrial DNA restriction profiles, most isolates could be classified into two distinct groups. These two groups could also be distinguished by the random amplified polymorphic DNA technique, and with telomeric PCR amplifications. Phylogenetic analysis of sequences of the intergenic transcribed spacer region of some of the strains resulted in a dendrogram with the same topology as that based on mitochondrial DNA and amplified DNA data. None of the isolates with type 2 mtDNA profiles produce ochratoxins. Some strains (e.g., A. ochraceus ICMP 939) displayed strain-specific mitochondrial DNA patterns, and their amplified DNA profiles were also different from all other A. ochraceus strains examined.

Aspergillus↗