Random Amplified Polymorphic DNA-polymerase Chain Reaction Analysis Research Articles

Molecular characterization and antimicrobial resistance of Enterococcus faecalis isolated from seafood samples.

Background Enterococcus faecalis is considered an opportunistic foodborne pathogen. The present study aimed to assess the prevalence, antimicrobial resistance, virulence characters, and molecular typing of E. faecalis strains isolated from seafood samples.MethodsTwo hundred and seventy‐six seafood samples were collected. E. faecalis was isolated from samples using bacterial culture. Furthermore, the disk diffusion assessed their antimicrobial resistance. Also, the distribution of virulence factors was determined using polymerase chain reaction (PCR) assay. Random amplified polymorphic DNA (RAPD) method was used for their molecular typing.ResultsFifty‐six of 276 (20.2%) seafood samples were contaminated with E. faecalis. Fish harboured the highest contamination rate (30.0%). Isolates harboured the highest resistance rate towards oxacillin (100%), tetracycline (100%), erythromycin (100%), cefoxitin (89.2%), cefazolin (87.5%), trimethoprim‐sulfamethoxazole (85.7%), rifampin (69.6%), clindamycin (69.6%), and gentamicin (64.2%) antimicrobials. Efa (100%), ebpA (89.2%), ebpB (58.9%), ebpC (53.5%), and esp (51.7%) were the most commonly detected virulence factors among E. faecalis isolates. RAPD–PCR analysis showed 11 different molecular clusters considering the closeness of more than 80%.ConclusionSeafood samples were considered reservoirs of virulence and resistant E. faecalis strains. Different molecular clusters of isolates may reflect their diverse sources of contamination.

Estimation of arsenic-induced genotoxicity in melon (Cucumis melo) by using RAPD-PCR and comet assays

In this study, arsenic (As)-induced genotoxicity in the roots and shoots of Cucumis melo (melon) seedlings were investigated by using the random amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR) and comet assays. For this purpose, melon seedlings were exposed to arsenate [As(V)] at 0, 100, 200, 300, and 400 ?M concentrations in the hydroponic system for 14 days to examine the level of As accumulation, alterations in growth parameters, and DNA damage. A reduction in growth with increasing As(V) concentration was observed in the melon seedlings. Total As accumulations in the shoot and root tissue increased in a dose-dependent manner; however, the level was higher in the roots than the shoots. In RAPD-PCR analysis, 26 primers gave reproducible and scorable results and produced a total of 128 bands in the control seedlings. Alterations in RAPD profiles, including the loss or appearance of new bands, were determined in the As-treated seedlings when compared to the control. The values of genomic template stability (GTS) were decreased by increasing the concentration of the As(V) in both tissue types. DNA strand breaks were observed in all the tested As(V) concentrations in the alkaline comet assay; furthermore, the loss of DNA integrity was higher with 300 and 400 ?M As(V) treatments. The results clearly indicate that the combination of DNA-based molecular and cytogenetic techniques (e.g. the comet assay) may be proposed as a reliable evaluation of genotoxicity in plants after exposure to heavy metal pollution.

Molecular characterization of Echinococcus granulosus in livestock of Al-Madinah (Saudi Arabia).

Echinococcus granulosus is the causative agent of cystic echinococcosis, which has serious impacts on human and/or animal health, resulting in significant economic losses. Echinococcus granulosus comprises a number of intra-specific variants or strains at the genetic level. In Saudi Arabia, few studies were performed on genetic variations in Echinococcus species. Therefore, the present study aimed to investigate the phenotypic and genetic characterization of hydatid cysts harboured by sheep and camels in Al-Madinah Al-Munawarah. Samples of hydatid cysts were collected from local sheep (n = 25) and camels (n = 8). The morphological criteria of protoscoleces were investigated. To investigate the molecular characterization, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR), single-stranded conformation polymorphism (SSCP) were carried out. DNA was extracted from individual fertile cysts and subjected to RAPD-PCR analysis (using five arbitrary primers) and PCR amplification of cytochrome c oxidase I (cox1) and 12S ribosomal ribonucleic acid (12S rRNA) genes. The PCR products were subjected to SSCP analysis for genetic discrimination in E. granulosus isolates. In addition, partially sequencing of the mitochondrial DNA cox1 genes was achieved for assessing the phylogenetic positions of collected isolates using some global published sequence data of cox1 genes. The rostellar hooks of camel and local sheep isolates show remarkable variability in their dimensions. Five distinct SSCP patterns were identified in the 12S rRNA gene, showing intraspecific variations in E. granulosus of camels and local sheep. Sequencing of (cox1) genes of both local sheep and camels exhibit high similarity with those of the same gene (E. granulosus sensu stricto) published in NCBI BLAST.

English

Limicolaria aurora belongs to the group of land snails commonly called garden snails. This study seeks to use shell morphology and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) to examine gross morphological differences in populations of L. aurora from New Bussa (guinea savannah) and Benin City (tropical rain forest) in Nigeria and possibly delimit the populations into sub species. A total of one hundred and ten specimens of L. aurora made up of fifty five individuals from each of the two ecological zones were collected randomly for the study. Data on shell parameters including: height of shell (SH), width of shell (SW), aperture height (AH), aperture width (AW), spire length (SL), and first whorl length (1WL) measured on each snail were subjected to one way analysis of variance (ANOVA). Principal component analysis (PCA) and canonical variates analysis (CVA) were performed on the data using PAST statistical software. DNA which was extracted from the muscular tissues of the foot of eight individuals from each location using cetyltrimethylammonium bromide (CTAB) method was subjected to RAPD-PCR. Amplification of the DNA was done using five primers (OPB-12, OPB-18, OPH-08, OPD-11 and OPS-13). Analyses showed significant differences (P<0.05) in L. aurora populations within and between the vegetation zones revealing great heterogeneity in the populations. Both PCA and CVA clusters did not separate the populations into distinct sub-populations. SH was the most variable morphological characteristic and consequently the most suitable for the separation of L. aurora specimens into distinct populations. All the primers used in the amplification of the DNA produced polymorphic bands. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster diagram revealed two major clades within the snail populations with about 74% similarity. The study showed that RAPD-PCR analysis is more suitable for delimiting populations of L. aurora than morphometrics and that the basis for gross morphological differences in these populations might not only be environmental but also genetic factors. Key words: Achatinidae, biodiversity, environmental factors, morphometrics, phylogenetics, shell, sub-populations, variation.

Assessment of Genetic Diversity among Newly Selected Roselle (Hibiscus sabdariffa Linn.) Genotypes in Nigeria Using Rapd-Pcr Molecular Analysis

In order to assess the genetic diversity among Nigerian Roselle (Hibiscus sabdariffa Linn.), certain genetic and morphological markers were used to group the various accessions collected into distinct genotypes. Molecular characterisation was then conducted on the twenty newly selected genotypes (NGR-HS-001, NGR-HS-002, NGR-HS-003, NGR-HS-004, NGR-HS-005, NGR-HS-006, NGR-HS-007, NGR-HS-008, NGR-HS-009, NGR-HS-010, NGR-HS-011, NGR-HS-012, NGR-HS-013, NGR-HS-014, NGR-HS-015, NGR-HS-016, NGR-HS-017, NGR-HS-018, NGR-HS-019, and NGR-HS-020) of the Roselle (H. sabdariffa Linn.), using Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) based techniques. The molecular characterisation was carried out at the Biotechnology Laboratory, International Institute of Tropical Agriculture (IITA), Ibadan. Five RAPD primers i.e. OPH05, OPB17, OPT17, OPB11 and OPB04 were able to amplify 30 reproducible fragments of which 100 % were polymorphic, indicating that Nigerian Roselle accessions are genetically heterogeneous; it also showed that RAPD analysis is an effective molecular marker in detecting genetic diversity among different Roselle genotypes in Nigeria. All the primers gave a high genetic diversity and high polymorphic information contents, however, OPT17 gave the highest genetic diversity and polymorphic information content (0.79 and 0.78 respectively). The Dice Dissimilarity index for the calculation of distances among the roselle accessions revealed an interesting phenomenon because a clear divergence among the H. sabdariffa accessions was found. The dissimilarity index ranged from 0-1, which revealed a wide range of genetic identity. Dissimilarity coefficient of accession NGR-HS-017 was the highest (1.00); this accession was totally different or genetically distance from all the other accessions. In addition, a high dissimilarity index (1.00) was also obtained among some of the accessions; NGR-HS-018 was highly dissimilar from NGR-HS-002, NGR-HS-012 and NGR-HS-016. However, a quite number of the accessions showed moderately low to zero dissimilarity indices. It is therefore concluded that RAPD-PCR analysis provided very valuable means for determining relationships among H. sabdariffa accessions. Wide genetic variability observed among the Roselle accessions could be exploited in different breeding programs of the crop. Therefore, other DNA markers like SSR and RFLP should be used to ascertain these levels of relationships.

Preliminary genetic diversity Analysis of introduced and local Zimbabwe cowpea landraces

Cowpea, Vigna Unguiculata plays an important role in human diets because of its good protein quality and high nutritional value. On hand are two cowpea lines sourced from a local Zimbabwean humanitarian organization, with an aim to diversify the local germplasm base of cowpea. Activities involved characterizing and evaluating the foreign sourced lines alongside local ones for morphological and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) diversity. Five seed characteristics were evaluated, namely seed size, color, uniformity, presence or absence of seed spots and hilum color using specified scales. For RAPD-PCR analysis, a set of five oligonucleotides was used. DNA extraction, quantification, amplification, electrophoresis and band analysis of presence or absence of bands was carried out. Data was collected and an ANOVA was carried out using genstat discovery software. After an ANOVA, principal components analysis (PCA) and hierarchical cluster analysis were used to examine distribution patterns in the germplasm. Significant differences were observed for the five variables (pE‚0.001). Both morphological and molecular data for the cowpea lines show limited diversity from results of multivariate analysis. The dendrogram for morphological data shows an overall similarity index of 78%, whereas for molecular data shows a similarity index of 75%. Of the two foreign lines, the white seeded foreign line, NGO1, seems to be consistently diverse from the rest. It will be a valuable addition to the collection and will be useful for possible introgression in future. The genetic diversity for the collection is generally low and may require continued enrichment with introductions in future. Key words: Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) characterization, variables, primers, electrophoresis, gel scoring.

Genetic diversity and identification of some Turkish cotton genotypes (Gossypium hirsutum L.) by RAPD-PCR analysis

Cotton (Gossypium hirsutum L.) is the leading fiber crop and the second most important oil seed crop in the world. This study aimed to analyze the genetic differences among 9 cotton genotypes of cultivars from Turkey using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. A total of 42 RAPD primers were used to determine polymorphism among the genotypes and 34 of the primers produced scorable results, with 319 RAPD-PCR bands obtained. The rate of polymorphism was calculated to be 18.1%, and genetic similarity between any 2 genotypes ranged from 90.2% to 96.5%. By evaluating the presence and absence of the RAPD-PCR bands, a dendrogram indicating the relative genetic similarity of the Turkish cotton genotypes was constructed and presented. The results also showed that the genotypes can be separated from each other at the molecular level by taking advantage of some of the RAPD markers. Specific markers were proposed for the identification of genotypes. To our knowledge, this is the first study to use RAPD markers to investigate the genetic relationships among the Gossypium genotypes grown in Turkey.

Use of autochthonous starters to ferment red and yellow peppers ( Capsicum annum L.) to be stored at room temperature

Strains of Lactobacillus curvatus, Leuconostoc mesenteroides, Lactobacillus plantarum and Weissella confusa were identified from raw red and yellow peppers (RYPs) by partial 16S rRNA gene sequence and subjected to typing by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis. L. plantarum PE21, L. curvatus PE4 and W. confusa PE36 were selected based on the kinetics of growth and acidification, and used as the autochthonous mixed starter for the fermentation of RYPs. A protocol which included blanching at 85 °C for 2 min, fermentation at 35 °C for 15 h in brine (1%, w/v), and heat treatment at 85 °C for 15 min, followed by storage at room temperature for 30 days with and without sunflower seeds oil was set up. Unstarted RYPs subjected to the same treatments were used as the control. Cell numbers of autochthonous starter in the RYPs were ca. 1000 times higher than presumptive lactic acid bacteria in unstarted RYPs. As shown by RAPD-PCR analysis, all three autochthonous strains persisted during processing and storage. Presumptive lactic acid bacteria found in started RYPs progressively decreased during storage, leading to a microbiota mainly consisting of autochthonous starters. Started RYPs showed rapid decrease of pH (< 3.7), marked consumption of fermentable carbohydrates, and inhibition of total enterobacteria and yeasts. Unstarted RYPs were subjected to slight acidification (pH ca. 4.87) and considerable contamination by total enterobacteria and yeasts throughout storage. After 30 days of storage, started RYPs had significantly ( P < 0.05) higher firmness and colour indexes with respect to unstarted RYPs. The microbial and sensory features of started RYPs stored with sunflower seeds oil were almost similar to those of RYPs stored without suspending liquid.

Characterization of two non-native invasive bark beetles, Scolytus schevyrewi and Scolytus multistriatus (Coleoptera: Curculionidae: Scolytinae)

AbstractScolytus schevyrewi Semenov, the banded elm bark beetle, and S. multistriatus Marsham, the smaller European elm bark beetle, are morphologically similar. Reliance on adult external morphological characters for identification can be problematic because of wide within-species variability and the need for good-quality specimens. The inability to identify developmental stages can also hamper early-detection programs. Using two character identification systems, genitalic (aedeagus) morphology, and DNA markers (random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)) to distinguish S. schevyrewi from S. multistriatus, we examined specimens from geographically distinct populations of both species collected from infested host trees or semiochemical-baited funnel traps. We found that aedeagus morphology can be used to identify the two species. The use of two oligonucleotide primers in the RAPD-PCR analysis yielded distinct DNA banding patterns for the two species. Species identification using RAPD-PCR analysis was validated by a blind test and used to make species identifications of larval specimens. These tools improve the ability to differentiate between S. schevyrewi and S. multistriatus at immature and adult stages, and could be developed and used for other scolytines as well.

Genetic Diversity within an Italian Population of ForestArmillaria gallicaIsolates as Assessed by RAPD-PCR Analysis

The genus Armillaria includes harmful fungal pathogens that cause root rot and wood decay in a broad range of host plants throughout the world. The aim of this study was to detect, by means of Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers, the level of intraspecific variability within isolates of an Armillaria gallica population sampled from a Quercus spp. stand located in Gravina in Puglia, southern Italy. UPGMA cluster analysis of RAPD profiles generated by decamer primers grouped the isolates in subclusters demonstrating relatively low intraspecific genetic variability. Moreover, RAPD pattern analysis yielded clusters which did not correspond to the groups discriminated by vegetative compatibility tests performed by a previous investigation on the same population. The findings of this research pose the question of whether somatic incompatibility, which involves an undefined number of genes and alleles per gene, might still be considered an effective tool for the epidemiological studies of A. gallica, whereas molecular analyses are more useful for assessing genomic variation within the species.

Spectrum of bacteriocin activity of Lactobacillus plantarum BS and fingerprinting by RAPD-PCR

The spectrum of antimicrobial activity of Lactobacillus plantarum BS against representative bacterial species was established through deferred assay and ‘spot-on-lawn’ assay using actively growing cells and partially purified bacteriocin extract, respectively. Only lactobacilli, pediococci, enterococci, bacilli and Listeria were inhibited from the test microorganisms. Slight bacteriocinogenic activity through ‘spot-on-lawn’ assay was detected against Staphylococcus aureus and Escherichia coli O157:H7. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis was used to compare the fingerprint of L. plantarum BS with other strains of L. plantarum. Using the 16S rRNA-based primer, P32, the bacteriocinogenic isolate exhibited identical RAPD-PCR fingerprints to L. plantarum ATCC 14917. Dendrograms derived from the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) were constructed to show the similarity relationships among the investigated strains based on RAPD-PCR analysis. Bands differentiating L. plantarum BS from L. plantarum ATCC 14917 were also identified by varying the annealing temperature.

Genotypic Identification of Saccharomyces Species using Random Amplified Polymorphic DNA Analysis

According to different molecular approaches the genus Saccharomyces was divided recently into 10 genotypically distinct species (S. bayanus, S. castellii, S. cerevisiae, S. dairensis, S. exiguus, S. kluyveri, S. paradoxus, S. pastorianus, S. servazzii, S. unisporus). This was corroborated by Random Amplified Polymorphic DNA — Polymerase Chain Reaction (RAPD-PCR) analysis in the present paper. Thirtytwo strains including the type strains of 20 Saccharomyces species defined originally by phenotypic characteristics (e.g. S. chevalieri, S. diastaticus, S. ellipsoideus) clustered with the pattern of S. cerevisiae, fourteen (e.g. type strains of S. globosus, S. heterogenicus, S. inustiatus) with the pattern of S. bayanus, six including the type strains of S. carlsbergensis and S. monacensis with the pattern of S. pastorianus and two with the pattern of S. paradoxus. Two further strains isolated newly were identified to belong to S. paradoxus. In comparison with nuclear DNA/DNA hybridization or electrophoretic karyotyping, RAPD-PCR anaylsis turned out to be a simple and reliable method to separate Saccharomyces species at the genotypic level. In contrast to phenotypic characters genotypic identification using RAPD-PCR analysis guarantees species specificity if type strains are included in the investigation. The ten Saccharomyces species arising from RAPD-PCR analysis are differentiated from each other to the maximal extent with exception of the relationship between S. bayanus and S. pastorianus. In this case, the estimated similarity value of 45% is significantly higher than the background noise (0–20%), but less than the values within species (83 to 100%).