Random-amplified Polymorphic DNA Polymerase Chain Research Articles

Random amplified polymorphic DNA-based molecular heterogeneity analysis of Salmonella enterica isolates from foods of animal origin.

Aim:This study aims to study the significance of random amplified polymorphic DNA (RAPD) typing in heterogeneity analysis of Salmonella serovars, isolated from foods of animal origin.Materials and Methods:Salmonella serovars isolated and identified from different foods of animal origin such as meat, milk, and egg by standard bacteriological methods. DNA isolated from all 10 isolates which are confirmed by biochemical and serotyping methods and then RAPD was performed using the primers OPB 10, primer 1290, NSC I, NSC II, and primer 3. Then, RAPD data were analyzed using the BioNumerics software, Belgium, Germany.Results:RAPD polymerase chain reaction (PCR) using five primers, namely OPB 10, primer 1290, NSC I, NSC II, and primer 3, classified the 10 isolates into 9, 10, 10, 7, and 10 RAPD-PCR types with discriminating powers of 0.1987, 0.423, 0.50889, 0.1842, and 0.2582, respectively. The phylogram constructed with NSC I profile classified isolates based on geographical origin. Primer 1290, NSC II, and primer 3 produced some uniform bands in all isolates indicating their binding ability in conserved genomic region. This study revealed that RAPD profile can be best used for finding out the heterogeneity at molecular level of Salmonella isolates in combination with other molecular and phenotypic typing techniques. Thus, our results support earlier observation of its significance by different workers on different Salmonella serotypes.Conclusion:Repeatability of RAPD-PCR is insufficient to distinguish genetic differences among Salmonella serovars.

DNA alterations and effects on growth and reproduction in Daphnia magna during chronic exposure to gamma radiation over three successive generations

This study examined chronic effects of external Cs-137 gamma radiation on Daphnia magna exposed over three successive generations (F0, F1 and F2) to environmentally relevant dose rates (ranging from 0.007 to 35.4mGyh−1). Investigated endpoints included survival, growth, reproduction and DNA alterations quantified using random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Results demonstrated that radiation effects on survival, growth and reproduction increased in severity from generation F0 to generation F2. Mortality after 21 days at 35.4mGyh−1 increased from 20% in F0 to 30% in F2. Growth was affected by a slight reduction in maximum length at 35.4mGyh−1 in F0 and by reductions of 5 and 13% in growth rate, respectively, at 4.70 and 35.4mGyh−1 in F2. Reproduction was affected by a reduction of 19% in 21 day-fecundity at 35.4mGyh−1 in F0 and by a delay of 1.9 days in brood release as low as 0.070mGyh−1 in F2. In parallel, DNA alterations became significant at decreasing dose rates over the course of F0 (from 4.70mGyh−1 at hatching to 0.007mGyh−1 after ∼21 days) and from F0 to F2 (0.070mGyh−1 at hatching to 0.007mGyh−1 after ∼21 days), demonstrating their rapid accumulation in F0 daphnids and their transmission to offspring generations. Transiently more efficient DNA repair leading to some recovery at the organism level was suggested in F1, with no effect on survival, a slight reduction of 12% in 21 day-fecundity at 35.4mGyh−1 and DNA alterations significant at highest dose rates only. The study improved our understanding of long term responses to low doses of radiation at the molecular and organismic levels in a non-human species for a better radioprotection of aquatic ecosystems.

POPULATION STRUCTURE OF THE POTATO TUBER MOTH ,PHTHORIMAEA OPERCULELLA (ZELLER) , LEPIDOPTERA: GELECHIIDAE) IN DELTA EGYPT USING RAPD ANALYSIS

Little is known about the nature and extent of genetic diversity in potato tuber moth Phthorimaea operculella (Zeller),Lepidoptera: Gelechiidae). Analysis of amplified fragment length polymorphism (AFLP) has the potential to become a powerfulnew DNA fingerprinting technique for studying genetic relationships and genetic diversity in insects. Random amplifiedpolymorphic DNA polymerase chain reaction (RAPD-PCR)was used to examine variation in three governorates in the Deltaof Egypt. The objective of this study was to provide baseline information on the population structure of potato tuber worm inEgypt. The applicability of CTAB -based DNA extraction protocol provided suitable for AFLP analysis. Potato tuber wormadults were collected from potato fields in the major production regions in the Egypt . We used 20 random 10-oligomer primerindividuals and 179 polymorphic AFLP bands were used to infer the geographic population structure of potato tuber worm.This study provides baseline data for the molecular characterization of potato tuber worm populations, which will aid in trackingthe origin of future invasions within Egypt.

Genetic diversity of Norwegian and Lithuanian red deer (Cervus elaphus) populations

During the last centuries, red deer (Cervus elaphus) have survived a drastic decline in population number which has gone as far as extinction in many regions of the continent. Today’s deer population is abundant and widespread, however, affected by re-introduction and keeping isolated animals in enclosures. It is necessary to observe the genetics of these animals and to monitor changes that occur in populations as well as apply the obtained knowledge in creating and restoring the species. This study investigates the levels of genetic diversity of red deer. Muscle and liver samples were obtained from 27 individuals corresponding to 2 red deer populations from Lithuania and Norway. Samples were analyzed by RAPD (random amplified polymorphic DNA) polymerase chain reaction (PCR) using ten primers (ROTH-180-01, ROTH-180-02, ROTH-180-03, ROTH-180-04, ROTH-180-05, ROTH-180-06, ROTH-180-07, ROTH-180-08, ROTH-180-09, ROTH-180-10). PCR products were sorted according to their size by electrophoresis in 1.7% agarose gel. Gel electrophoresis showed that there were 64 polymorphic loci with the fragment size varying from 150 to 3 000. The “TREECON for Windows” programme was used for the estimation of genetic variability. Genetic variability among the red deer separate individuals varied from 0.08 to 0.80. Keywords: red deer (Cervus elaphus), RAPD, PCR, ROTH-180, genetic diversity

Elucidation of genetic variability among different isolates of Fasciola gigantica (giant liver fluke) using random-amplified polymorphic DNA polymerase chain reaction

The present communication focuses on molecular characterization of Fasciola gigantica isolates derived from cattle, buffalo, and goat using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) analysis to elucidate genetic variability between the three isolates. Seventeen random oligonucleotide primers of 10-11 bases with GC content varying from 50-81.8% were used in the study. Depending upon the F. gigantica isolate-primer combination, one to five fragments in the range of 327-1,973 bp were amplified. It was significant to observe that, out of the 17 primers directing amplification of DNA fingerprints, only two designated as AP9 and AP14 were found to be of potential interest in the generation of polymorphic DNA. On the basis of similarity coefficient data, it may be suggested that cattle and buffalo isolates of F. gigantica show 100% homogeneity against 92.68% similarity coefficient observed between goat and cattle/buffalo isolates. In other words, 7.32% divergence was observed between goat and cattle/buffalo isolates while the primers AP1, AP4, AP10, AP13, and AP17 were able to generate monomorphic DNA fingerprints. Primers AP9 and AP14 are potentially informative in terms of the polymorphic nature of the fingerprints generated in RAPD assays. The finding of the absence of 626-bp DNA fragment in the goat and the uniqueness of the AP14 in generating a single RAPD-PCR product of 1,211 bp as against the product size of 1,162 bp in cattle/buffalo seem to be significant. This is the first report of elucidation of RAPD-PCR based molecular variability in the DNA fingerprinting pattern of F. gigantica isolated from cattle, buffalo, and goat.

Genetic variability in Daphnia magna and ecotoxicological evaluation

The fresh water crustacean Daphnia magna is widely used as a test organism in aquatic toxicology to assess the adverse effects of individual substances or complex mixtures, e.g. industrial wastewaters. Cultures are held in several European testing laboratories and testing is typically carried out according to internationally standardised protocols. However, despite accounting for many potential confounding factors these guidelines do not currently take into account any specification related to the use of a specific clone. Cultures from seven laboratories were used to assess genetic variability by random-amplified polymorphic DNA polymerase chain reaction. Results pointed out the existence of two main clone clusters Responses in the acute Daphnia immobilisation test showed no direct correlation with genetic clusters resulting from random genetic markers (random-amplified polymorphic DNA) analysis. Considering that genetic differences are the most probable cause for the ecotoxicological test data, further analysis concerning gene expression and genetic stability should be performed.

An outbreak of Serratia marcescens on the neonatal unit: a tale of two clones

Serratia spp. are an important cause of hospital-acquired infections and outbreaks in high-risk settings. Twenty-one patients were infected or colonized over a nine-month period during 2001-2002 on a neonatal unit. Twenty-two isolates collected were examined for antibiotic susceptibility, beta-lactamase production and genotype. Random-amplified polymorphic DNA polymerase chain reaction and pulsed-field gel electrophoresis revealed that two clones were present. The first clone caused invasive clinical infection in four babies, and was subsequently replaced by a non-invasive clone that affected 14 babies. Phenotypically, the two strains also differed in their prodigiosin production; the first strain was non-pigmented whereas the second strain displayed pink-red pigmentation. Clinical features suggested a difference in their pathogenicity. No environmental source was found. The outbreak terminated following enhanced compliance with infection control measures and a change of antibiotic policy. Although S. marcescens continued to be isolated occasionally for another five months of follow-up, these were sporadic isolates with distinct molecular typing patterns.

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.

Application of RAPD and cytochrome b sequences to the study of genetic variations in F1 and F2 generations of two different strains of guppy (Poecilia reticulata, Peter 1854)

The DNA of two laboratory strains of guppy (Poecilia reticulata), Black Yellow (BYS) and Red Flame (RFS), was studied with respect to their colour differences, in two generations (F1 and F2). Their varying morphological colours were related to their cloned fragments of cytochrome b mitochondrial DNA (mtDNA) and random-amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The F1 generation was characterized by various forms. The male BYS could be divided into 68% having black–yellow tails and 14% having black–yellow–red tails. The other variations were found in very low percentages. The percentage of black–yellow-tailed males increased to 85 in the F2 generation, and the percentages of the various other forms consequently decreased. Only 63% of BYS males had black–yellow dorsal fins in the F1 generation, but this percentage increased to 84 in F2. Compared with the males, fewer variations were found in female colour patterns in the F1 generation. A high percentage of BYS females (81%) colour was found with no significant increase in the F2 generation. However, variations decreased in the F2 females. On the other hand, a very high variation was found in female fins in the F1 generation: only 32% were of BYS colour and 25% had no-colour fins. However, a significant increase in BYS colour was found in the F2 generation (61%) and 39% had no colour. The variation in RFS was lower than BYS in the F1 generation: 81% of the F1 males had red–yellow tails with colour, 46% of the fins were yellow and 36% were red–white. In females, a very high percentage (84%) had red–yellow tails and 76% had no-colour dorsal fins. Mitochondria DNA markers and genomic DNA were studied in various laboratory strains. In the clone of the fragments of cytochrome b, the bands correlated to the colour phenotype. A fragment of the cDNA sequence was determined from a 268-bp cloned with fragments of the guppy cytochrome b mtDNA gene. The genes varied for the two strains in only two base pairs, starting at the nucleotide position 171 and ending at position 174. Three primers showed good results in the RAPD-PCR and were found suitable for the study of DNA variations in guppy. The high variation detected in BYS, in comparison with RFS, was reflected by changes in band-sharing (BS) values ranging from 0.66 to 1, versus 0.8 to 1 in RFS.

Detection of Hammondia heydorni-like organisms and their differentiation from Neospora caninum using random-amplified polymorphic DNA-polymerase chain reaction.

Neospora caninum and Hammondia heydorni are morphologically and phylogenetically related coccidians that are found in dogs. New diagnostic genetic loci, based on random-amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), were developed to aid in the detection of H. heydorni-like parasites and to discriminate them from N. caninum and other related coccidians of dogs. On the basis of the data obtained from 5 random decamers, H. heydorni (Manhattan-1) and N. caninum (NC1) were characterized by distinct banding patterns (similarity index = 0.068). High-stringency PCR assays were developed from the sequences of 2 cloned bands (GenBank BZ592549 and BZ592593), uniquely amplified from H. heydorni. Interestingly, using these primers, PCR amplification was achieved only from 2 of the 5 isolates presumed to represent H. heydorni. The same result was obtained from these 5 isolates using a recently described PCR assay directed to the H. heydorni internal transcribed spacer-1. It is concluded that H. heydorni and N. caninum are genetically distinct and that such tools may be useful for more detailed characterization of the diversity of related parasites occurring in dogs.

Genetic polymorphism in nine clones of a parthenogenetic collembolan used in ecotoxicological testing

Ecotoxicological tests are increasingly being used to evaluate the toxicity of a substance or mixture of substances towards soil fauna. One of these uses the parthenogenetic collembolan Folsomia candida to test for soil contamination, and this has been standardised in Europe. However, in such studies, it is important to know whether different laboratories use genetically close clones. In this study, nine clones of the parthenogenetic collembolan F. candida, used in five laboratories, have been subtyped by amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). This differentiated these clones into three groups of four, four and one, respectively. Several hypotheses can be envisaged to explain these divergences between the populations of this parthenogenetic collembolan, notably the history of interlaboratory exchanges of clones. Ecotoxicological tests (mortality and reproduction) were undertaken to test the sensitivities to cadmium and phenantrene of two clones from different RAPD defined groups. These showed minor differences in sensitivities to both, but more extensive testing with five cadmium concentrations showed that the genetic clone does not affect the LOEC determination. There is no evidence of significant differences between clones throughout a concentration gradient of cadmium.

Genomic differences in Streptococcus pyogenes serotype M3 between recent isolates associated with toxic shock-like syndrome and past clinical isolates.

Genomic differences among past Streptococcus pyogenes serotype M3 strains isolated in 1973 and before from patients with streptococcal pharyngitis, recent (1990s) serotype M3 clinical isolates from patients with pharyngitis, and recent M3 isolates from patients with toxic shock-like syndrome were investigated by restriction landmark genomic scanning and by modified random-amplified polymorphic DNA-polymerase chain reaction. Similar polymorphic DNA fragments were identified between the older M3 isolates and the recent isolates; also, the recent M3 clinical isolates from patients with pharyngitis were genetically indistinguishable, by the methods used, from the M3 isolates of patients with toxic shock-like syndrome. Although nucleotide sequences of these regions showed no apparent homology with known virulence factors, the DNA fragments could distinguish the recent M3 strains from the past strains. These results suggested that the recent strains have emerged because of genetic divergence.

Identification and characterization of a new group of root‐colonizing fungi within the Gaeumannomyces–Phialophora complex

A new group of darkly pigmented root‐infecting fungi was isolated from cereal roots obtained from six different locations in northeastern Germany. Similar random amplified polymorphic DNA(RAPD) patterns and restriction profiles of amplified rDNA were used as a basis for classifying the isolates in a separate group. The isolates demonstrating mycelial and infection characteristics typical of Gaeumannomyces graminis could be differentiated from the varieties of G. graminis as well as from Gaeumannomyces cylindrosporus/Phialophora graminicola using RAPD Polymerase chain reaction (PCR) and rDNA Restriction‐fragment length polymorphism (RFLP) analysis. Phylogenetic analysis of the Internal transcribed spacer (ITS) regions suggests that the isolates form a distinct group (named group ‘E’) situated within the Gaeumannomyces–Phialophora complex between the branch of the G. graminis varieties and Gaeumannomyces incrustans/Magnaporthe poae. Isolates of group E produced lobed hyphopodia and were shown in biotests to be non‐pathogenic to wheat, oats, Italian Ryegrass and Chewings Fescue, suggesting it is a benign parasite which colonizes cereals or grasses without destroying vascular tissue. Furthermore, curved phialospores could be found. Summarizing the results presented, this new group could be classified as a new species of Phialophora. Although isolates of group E were found at only six of the 32 investigated locations, they composed up to 50% of total isolates of the Gaeumannomyces–Phialophora complex at these sites. Because of the non‐pathogenic behaviour, the new group may be of value as biological control agents for pathogenic fungi.

Temporal Changes in the Population Genetics of Salmonella pullorum

Salmonella pullorum is the cause of pullorum disease, which is characterized by white diarrhea and a high mortality rate in poultry. During the 1990s, the serologic "pullorum" test has occasionally failed to detect infected birds during the early stage of disease. To determine if any recent genetic changes have taken place in S. pullorum to account for poor seroconversion sometimes observed in infected flocks, S. pullorum from 1990s outbreaks and strains isolated prior to the 1980s were typed by random amplified polymorphic DNA (RAPD). Of 40 S. pullorum isolates typed by this method, eight distinct DNA patterns were identified with one of three RAPD polymerase chain reaction primers. Sixty-two percent of S. pullorum isolates shared the same RAPD DNA pattern, and a major proportion of these strains were from recent flock infections. The RAPD patterns for S. pullorum were clearly distinct from the avian Salmonella group B isolates included in this analysis. The distribution of Salmonella virulence genes among avian Salmonella isolates was also examined. Eighty-five percent of the S. pullorum isolates had both the virulence plasmid gene, spvB, and the invasion gene, invA, with the same percentage positive for the Salmonella enteriditis fimbrial gene, sef. However, significant variability was observed among S. pullorum in their ability to invade avian epithelial cells, despite the presence of the Salmonella invasion gene in these isolates.

Freeze Tolerance, Morphology, and RAPD-PCR Identification of Trichinella nativa in Naturally Infected Arctic Foxes

Arctic foxes (Alopex lagopus) were collected from Greenland and Svalbard (N = 319). Twenty-four were infected with Trichinella (7.5%). Molecular analysis (random-amplified polymorphic DNA polymerase chain reaction) confirmed that all animals were infected with Trichinella nativa. Motile larvae were found in muscle tissue from all foxes after carcasses had been frozen for 1 yr at -18 C. Infective larvae were found in 2 foxes after a total of 4 yr storage at -18 C, which is longer than any previous observations. Morphological examination of the cysts showed large nurse cells and significant deposition of collagen and connective tissue. It is suggested that, within the geographical distribution of T. nativa, the more freeze-resistant isolates are found at higher latitudes.

Species Recognition of Congeneric Acanthocephalans in Slider Turtles by Random-Amplified Polymorphic DNA (RAPD) Markers

Species recognition of acanthocephalans of the genus Neoechinorhynchus (Hamann, 1892) found in the freshwater turtle Trachemys scripta (Wied, 1838) has previously been based primarily on female body and egg morphology. Observed morphological plasticity within and among species may lead to the misclassification of female specimens and leaves males of different species completely indistinguishable. Here, random-amplified polymorphic DNA (RAPD) analysis was used to genetically characterize samples of Neoechinorhynchus pseudemydis (Cable and Hopp, 1954), Neoechinorhynchus emydis (Leidy, 1851), and Neoechinorhynchus emyditoides (Fisher, 1960). Amplifications performed with 3 decamer oligonucleotides showed banding patterns with a few diagnostic fragments that allowed the recognition of N. pseudemydis specimens from those of the N. emydis-N. emyditoides group. No primer gave a species-specific locus that allowed the differentiation of N. emydis from N. emyditoides specimens, suggesting that they could belong to a sole taxon. The species assignment of females of uncertain classification and of males is fully reliable using RAPD markers. Thus, identification of acanthocephalan species by RAPD in the helminth infracommunities could potentially be very useful to determine community structure. RAPD and other polymerase chain reaction-based methods have some practical advantages over multilocus discriminant analysis, such as the ability to use ethanol-stored specimens and small tissue samples of parasites.

Identification and Differentiation of Brewing Yeasts Using Specific and RAPD Polymerase Chain Reaction

Specific polymerase chain reaction (PCR), using oligonucleotide primers that target the intervening region between the 5s and 16s rRNA genes, was used to differentiate commercially important brewing Saccharomyces cerevisiae variants from other yeast. In addition, random amplified polymorphic DNA (RAPD)-PCR with short oligonucleotide primers was used to identify yeast variants within S. cerevisiae. The fingerprint patterns that were generated by the RAPD-PCR technique could also be used to differentiate between brewing and non-brewing yeast. It was observed using this technique and the respective primers that ale yeast DNA was a better target for primer annealing than was lager yeast DNA.

Use of genetic polymorphisms detected by the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations.

Amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to differentiate and identify mosquito populations based on genetic variation. Genomic DNA was extracted from individual mosquitoes from 11 geographic populations of Aedes aegypti and amplified in PCR reactions using single primers of arbitrary nucleotide sequence. Discriminant analysis of the population frequencies of RAPD fragments produced using three different primers allowed accurate discrimination between the geographic populations in 89% of individuals and between subspecies (Ae. aegypti aegypti versus Ae. aegypti formosus) in 100% of mosquitoes tested. The genetic relatedness of the populations was estimated using three different statistical methods, and unknown populations were correctly classified in a blind test. These results indicate that the RAPD-PCR technique will be useful in studies of arthropod molecular taxonomy and in epidemiologic studies of the relatedness of geographic populations and vector movement.