Random Amplified Polymorphic DNA Profiles Research Articles

Epidemiology and molecular genotyping of echinostome metacercariae in Filopaludina snails in Lamphun Province, Thailand

ObjectiveTo analyze the prevalence of echinostome metacercariae inFilopaludina dorliaris (F. dorliaris) and Filopaludina martensi martensi (F. martensi martensi) and genotype variation of echinostome metacercariae by using random amplified polymorphic DNA (RAPD) analysis. MethodsFilopaludina sp. snails were collected from eight localities of Lamphun Province, Northern Thailand and examined for echinostome metacercariae. RAPD-PCR was used to analyze genotype variation of echinostome metacercariae. ResultsA total of 3 226 F. dorliaris and F. martensi martensi snails were collected from eight localities. The overall prevalences of echinostome metacercariae in F. dorliaris and F. martensi martensi were 40.89% and 36.27%, while the intensity of infection was 20.37 and 12.04, respectively. The dendrogram constructed base on RAPD profiles, 4 well supported domains were generated; (i) group of metacercariae from Ban Hong, Mae Ta, Meaung, Pa Sang, Toong Hua Chang, and Weang Nong that were clustered in the group of E. revolutum, (ii) Ban Thi, (iii) Lee, and (iv) 3 adults of an out group. ConclusionsThis research demonstrated RAPD profiling has been a useful tool to detect DNA polymorphisms to determine genetic relationship between echinostome metacercariae in Lamphun Province, Northern Thailand.

<i>Escherichia coli</i> Population-Based Study in Pediatric Crohn’s Disease

Escherichia coli is assumed to be involved in inflammatory bowel disease (IBD) by many authors. The present Short Report was aimed at analyzing E. coli population isolated from ileal samples collected from 4 CD and 3 non-CD (control group) diagnosed pediatric patients. A total of 539 mu- cosa-associated E. coli strains were characterized by: Random Amplified Polymorphic DNA (RAPD), adhesive and virulence factors, and for their phylogenetic groups. A significant separation be- tween RAPD profiles of the two CD and non-CD cohorts (P < 0.0001), along with a significant reduc- tion of intra-species genomic variability in E. coli populations isolated from CD group (P < 0.0001) was found as assessed by Dice index, indicating a different selective pressure in CD intestinal ha- bitat. A predominance of phylogenetic group A was found in control subjects (P < 0.0001). Results on adhesive and virulence factors evidenced peculiar genes significantly related to CD E. coli pop- ulations (K1, IbeA) (P < 0.0001) and to total DNA from biopsy specimens K1 (P < 0.0001). Results suggest that among E. coli population, particular variants may be favorite in the intestinal habitat of CD pediatric patients. These genotype variants could represent the genetic background that, via evolutionary phenomena driven by a persistent inflammatory state, may evolve in Adherent Inva- sive Escherichia coli (AIEC) like strains found in adult CD patients.

The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia

Background: Random Amplified Polymorphic DNA (RAPD) assay has recently emerged as a genotyping method which is both robust and highly discriminatory for bacterial strain differentiation. However, RAPD assessment for Mycobacterium tuberculosiscomplex (MTC) isolates is still limited. Despite its simplicity and rapidity, conventional RAPD also has low reproducibility due to its sensitivity to several factors. Therefore we studied the feasibility of an RAPD-PCR assay to define the genetic diversity of MTC isolates and to evaluate its reproducibility.Methods:493clinical MTC isolates from the island of Sulawesi in Eastern Indonesia, collected from 2005-2012were subjected to Multiplex RAPD assay using 11 random decamer primers instead of one primer which is common in conventional RAPD. All 11 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for MTC. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers.Results:Multiplex RAPD-PCR results show that the majority of the isolates from South Sulawesi, Southeast Sulawesi andCentral Sulawesiin eastern region of Indonesia belong to group MT-C (80.7%, 80.0% and 62.6 % respectively) with result reproducibility as high as 100%. Conclusion:Molecular typing with multiplex RAPD-PCR is a powerful approach to show the genetic heterogeneity of MTC isolates. The discrimination power and reproducibility of this multiple loci-based RAPD was higher than conventional fewer loci-targeted RAPD.

Assessment of genetic stability of olive in vitro propagated by RAPD marker

The effect of sub culturing frequencies and different cytokinins (benzyl amino purine (BAP), 2-isopentenyl adenine (2ip) on genetic stability of micropropagated shoots from olive plant were investigated by using physiological traits and Random Amplified Polymorphic DNA (RAPD) markers. Axillary buds of the olive (cv. Dezful) plants were cultured and subcultured in DKW medium, supplemented with BAP (4 mgl-1) or 2-ip (4 mgl-1). In different hormone media and subcultures there were not significant differences in shoot proliferation rate. To amplify DNA, 18 arbitrary decamer primers were screened, out of which 16 primers generated clear and reproducible bands. All RAPD profiles from the micropropagated plants were monomorphic. The treatments included four successive subcultures in two hormone treatments (2ip or BAP) (4 mgl-1) The 16 primers produced a total of 213 (an average of 13.31 band per primer) scorable bands. The dendrogram constructed on the basis of jaccard’s similarity matrix, followed by UPGMA based clustering analysis showed that micropropagated plants were genetically stable and similar to the mother plant.

DNA polymorphism analysis of Xanthomonas campestris pv. campestris using single-strand conformation polymorphism (SSCP) and random amplified polymorphic DNA (RAPD)

The applicability of the random amplified polymorphic DNA (RAPD) and single-strand conformation polymorphism (SSCP) techniques using M13 and 16S rRNA primers, respectively, for genotyping of the phytopathogenic bacterium Xanthomonas campestris pv. campestris was studied. RAPD provided a simple, rapid, and reliable method to identify genetic variation, and SSCP was used to screen for sequence polymorphisms. A 730-bp region amplified using 16S rRNA primers was subjected to restriction digestion followed by SSCP analysis (restriction fragment length polymorphism (RFLP)-SSCP) to identify polymorphisms, and the results were compared with the RAPD profile obtained using the phage M13 core sequence as a single primer. SSCP is a useful tool for the detection of mutations but large amplicon size can hinder secondary structure formation. Therefore, RAPD using an M13 primer is more efficient for phylogeny detection. The use of a novel polymerase chain reaction (PCR)-based DNA fingerprinting method using both RAPD and SSCP to detect DNA sequence diversity is reported. This approach consists of PCR amplification of the 16S ribosomal DNA with universal primers and analysis of the PCR product by SSCP. This is the first report wherein plant pathogens are subjected to RFLP-SSCP combined with M13 primer-based analysis. This report provides a rapid and reliable method for phylogenetic analysis. Keywords: Xanthomonas campestris pv. campestris, polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), microbial phylogenetics, randomly amplified polymorphic DNA (RAPD). Afr. J. Biotechnol. Vol. 12 No. 50

English

A study to compare the effectiveness of inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) profiling was carried out with a total of 65 DNA samples using 12 species of Indian Garcinia. ISSR and RAPD profiling were performed with 19 and 12 primers, respectively. ISSR markers generated a total number of 156 bands with 92 polymorphic bands, while RAPD markers produced a total of 134 bands with 80 polymorphic bands. Percentage of polymorphic loci in RAPD profiling was 60.4% while in ISSR profiling, it was 59.3%. Heterogeneity index was similar for the markers, 0.86 for ISSR and 0.89 for RAPD, indicating that both the marker systems are effective in determining polymorphism in Garcinia. ISSR markers showed clear distinction among the species whereas RAPD markers showed segregation based on geographical location as well as species based. &nbsp; Key words: Garcinia, genetic diversity, inter-simple sequence repeats, randomly amplified polymorphic DNA, principal component analysis.

RAPD analysis of genetic diversity among the isolates of Aspergillus flavus from different hosts and locations

Aflatoxin contamination is a major problem in maize, groundnut, chillies, cotton and tree nuts. These aflatoxins are low molecular weight toxic and carcinogenic secondary metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. In the present study, a total of 11 isolates of A. flavus isolated from groundnut, maize and chilli collected from different locations of Tamil Nadu, India were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay. The results show that the isolates vary in their level of toxin production. The amount of AFB1 produced by the toxigenic isolates of A. flavus ranged from 6.6 to 108.1 ng ml−1. Among the various isolates of A. flavus, the isolate VKR produced the highest amount (108.1 ng ml−1) of AFB1. The isolates viz. CBE1, CBE2, BSR1, BSR3 and BSR4 were found to be non-toxigenic. The genetic variability among these isolates was assessed by Random amplified polymorphic DNA (RAPD) analysis. DNA fragments of between 0.15 and 3.0 kb were obtained using 13 random primers, and each isolate differed in the size and number of PCR products indicating considerable polymorphism. Cluster analysis using Unweighted Pair Group Method with Arithmetic Mean clearly separated the isolates into four main clusters confirming the genetic diversity among the isolates of A. flavus. Both toxigenic and non-toxigenic isolates were intermingled in these four groups, indicating that no relationship exists between RAPD profile and the production of aflatoxin by A. flavus.

English

Genetic structure of four populations of Mugil cephalus from Gujarat, Maharashtra, Andhra Pradesh and Tamil Nadu in India was studied using randomly amplified polymorphic DNA (RAPD) markers. Five selective primers provided distinct and consistent RAPD profiles in all the four populations. The bands in the range 400 to 1200 bp were scored for consistent results. The RAPD profiles generated by all the five primers revealed varying degrees of polymorphism, ranging from 50.76 (primer E03) to 72.41% (primer E05). Nei’s genetic diversity (h) among the four populations varied from 0.3717 ± 0.1460 (Gujarat population) to 0.5316 ± 0.1720 (Maharashtra population). Nie’s highest genetic distance (0.8556) was observed between Tamil Nadu and Gujarat populations.

English

We optimized the conditions for generating random amplified polymorphic DNA (RAPD) profiles of Streptococcus thermophilus strains by using the polymerase chain reaction (PCR). Several factors can cause the amplification of false and non reproducible bands in the RAPD profiles. We tested three primers, OPI-02 MOD, M13 and XD9 throughout this study. In addition, we tested different concentrations of primer, DNA template and Taq DNA polymerase. We adjusted the ratio of the primer to DNA template. All the three primers yielded reproducible profiles on several days, under optimized concentrations of components and cycling parameters used. The bands of such profiles probably corresponded to perfect annealing sites amplified with good efficacy or present in multiple copies in the genome. Five months later, repeated experiment generated identical bands. However, extra faint bands were detected with M13 and XD9 primers, possibly, corresponding to nonspecific binding resulting from slight variation in temperature or calibration of the thermocycler. Therefore, OPI-02 MOD was determined as the most reliable primer for reproducible profiles of S. thermophilus strains. &nbsp; Key words: Streptococcus thermophilus, random amplified polymorphic DNA (RAPD), DNA template, Taq DNA polymerase, OPI-02MOD, XD9, M13, optimization, reproducibility.

Molecular characterization of Candida spp. isolates from patients with bloodstream infections

The aim of this study was to conduct an epidemiological study comparing the genetic similarity of yeasts isolated from blood cultures. Random amplification of polymorphic DNA (RAPD) techniques were used for the Candida samples obtained from patients at the Hospital Universitário da Universidade Federal do Mato Grosso do Sul (HU/UFMS) in Campo Grande, State of Mato Grosso do Sul, Brazil, from 1998-2000. The most frequently isolated species was Candida albicans (45.8%). DNA amplification from genomic yeast isolates indicated a genetic similarity of over 90%. The RAPD profiles obtained were able to differentiate between the isolated Candida species, thereby suggesting that the method might be useful in epidemiological studies.

Characterization and antimicrobial resistance analysis of avian pathogenic Escherichia coli isolated from Italian turkey flocks

This study investigated the occurrence of avian pathogenic Escherichia coli (APEC) in a finishing turkey commercial farm, carrying out longitudinal surveys involving 3 consecutive flocks. The diversity and the distribution of the E. coli strains detected during colisepticemia outbreaks were examined. The strains were isolated, serogrouped, assessed for the presence of virulence-associated genes, typed by random amplification of polymorphic DNA (RAPD), and antimicrobial resistance analysis was then carried out. Escherichia coli O78 and O2 were predominantly found. Moreover, based on the somatic antigens used in the study, strains were recovered that were nontypeable. On one occasion, an E. coli O111 strain was found in turkeys. The E. coli isolates differed in terms of antibiotic resistance and RAPD profile. All strains possessed the virulence genes that enabled them to be considered APEC. Strains not only differed between flocks, but also within the same flock. These findings point out the importance of addressing colibacillosis therapy on the basis of a sensitivity test.

Mineral nutrient imbalance, total antioxidants level and DNA damage in common bean (Phaseolus vulgaris L.) exposed to heavy metals.

The present study aimed to analyze the biological effects induced by bioaccumulation of metals in common bean (Phaseolus vulgaris L.). Effects of mineral nutrient imbalance, total antioxidants level and DNA damage induced by accumulation of heavy metals, were investigated in bean seedlings treated with two selected metal concentrations for 7days. Metal content is analyzed by inductively coupled plasma - atomic emission spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay is used and Random Amplified Polymorphic DNA (RAPD) method was applied for investigation of DNA damages. The increasing metal concentration in the treatment medium changed synchronously metal content in samples, and decreased total antioxidant activity in all samples with exception only for samples treated with Ni and Cd. The obtained "DNA fingerprints" demonstrated that the increasing metal concentrations induced changes in RAPD profiles (disappearance and/or appearance of bands in comparison with untreated control samples). The highest number of missing bands was observed in samples treated with zinc (total 4 bands) and nickel (total 4 bands) at both concentrations. These results suggested that mineral nutrient imbalance is involved in changes of antioxidant levels and DNA damages of the seedlings, which may help to understand the mechanism of metal toxicity in plants.

In vitro propagation and assessment of clonal fidelity of Nepenthes khasiana Hook. f.: a medicinal insectivorous plant of India

An efficient in vitro protocol for large-scale multiplication of Nepenthes khasiana, a threatened insectivorous plant of India, has been developed from nodal stem segments. The highest shoot proliferation of 19.16 ± 0.23 shoots/explant was recorded in half-strength Murashige and Skoog (MS) medium supplemented with 2.5 mg/l kinetin, 2.0 mg/l 6-benzyl aminopurine, 3 % sucrose and 0.8 % agar. The best rooting was achieved in half-strength MS medium supplemented with 2.0 mg/l α-naphthalene acetic acid with an average of 9.04 ± 0.46 roots/shoot. The plantlets were successfully transferred to the greenhouse with survival rate of 92 %, exhibiting normal development. Cytological and random amplified polymorphic DNA (RAPD) analyses were carried out to assess the genetic integrity of the regenerated plantlets. Cytological analysis revealed no change in chromosome number with cells studied showing 2n = 80. Of the 80 primers screened for RAPD analysis, 14 primers resulted in clear and scorable bands. A total of 72 amplification products were obtained out of which only 4.1 % bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient ranging from 0.98 to 1.0, thus suggesting genetic stability in the micropropagated plants of N. khasiana.

Determination of genotoxic effects of boron and zinc on Zea mays using protein and random amplification of polymorphic DNA analyses.

In this research, we aimed to determine genotoxic effects of boron (B) and zinc (Zn) on Zea mays by using total soluble protein content and random amplification of polymorphic DNA (RAPD) analyses. For the RAPD analysis, 16 RAPD primers were found to produce unique polymorphic band profiles on treated maize seedlings. With increased Zn and B concentrations, increased polymorphism rate was observed, while genomic template stability and total soluble protein content decreased. The treatment with Zn was more effective than that of B groups on the levels of total proteins. The obtained results from this study revealed that the total soluble protein levels and RAPD profiles were performed as endpoints of genotoxicity and these analyses can offer useful biomarker assays for the evaluation of genotoxic effects on Zn and B polluted plants.

Assessment of paraquat genotoxicity on barley (Hordeum vulgare L.) seedlings using molecular and biochemical parameters

The effects of exposure of barley seedlings to different concentration (10−6 to 10−3 mol/l) of paraquat on seed germination, root length, antioxidant enzyme activities and randomly amplified polymorphic DNA (RAPD) profiles were investigated. The results revealed that malondialdehyde content significantly increased by exposing paraquat in a concentration-dependent manner (p < 0.05). A significant increase in peroxidase and catalase activities in seedlings was observed with increased concentration of paraquat, and then decreased when the value reached 10−3 mol/l, whereas the activities of superoxide dismutase gradually increased with increasing paraquat concentration. Germination and root elongation also decreased with the increase of paraquat concentration. On the other hand, alterations of DNA in barley seedlings were detected using RAPD technique. The changes occurring in RAPD profiles of seedlings following paraquat treatment included loss of bands found in DNA of control seedlings and appearance of new bands. The results of this study showed that paraquat induced DNA damage in a dose-dependent manner and the root cells of barley showed a defense against paraquat-induced oxidative stress by enhancing their antioxidant activities.

Phylogenetic character mapping of RADES Probing, a new marker for exploring the clonal evolution of expressed coding sequences in Trypanosoma cruzi, the agent of Chagas disease

We have tested a new genetic marker, RADES Probing (RADES-P), on a standard sample of 19 laboratory-cloned stocks of Trypanosoma cruzi, the agent of Chagas disease. This set of stocks, fully characterized using multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD), is representative of this parasite’s main genetic subdivisions. RADES-P consists in hybridizing RAPD profiles with probes composed of the products of random amplified differentially expressed sequences (RADES). The profiles thus obtained uncover only expressed coding sequences that are as well present on RAPD gels. Direct visual examination and the banding record show that these RADES-P profiles are different of, and not redundant with, both RAPD and RADES patterns obtained on the same set of stocks with the same primers. Phylogenetic character mapping (PCM) of the RADES-P polymorphism fairly confirms the known population structure and phylogenetic diversity of T. cruzi. This suggests that the impact of clonal evolution on T. cruzi has been predominant enough over the long term to carve the polymorphism of all types of DNA sequences, including polymorphisms of expressed coding sequences, although these sequences are subject to natural selection.

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity.

The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments, and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help in monitoring seed deterioration. Dry seeds were subjected to artificial ageing and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (random amplified polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin-Ciocalteu reagent method. Electron paramagnetic resonance (EPR) provided profiles of free radicals. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (type 2 metallothionein) and SOD (superoxide dismutase). A modified QRT-PCR protocol was used to determine telomere length. The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial ageing. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterized by a lower antioxidant specific activity. Significant upregulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non-enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species.

Impact Assessment of Diesel Oil on the Zhe Oyster (Crassostrea plicatula) Using RAPD Analysis

The lethal and sublethal effects of oils on aquatic organisms have been widely investigated, but the potential molecular impacts of oils on aquatic organisms are remaining unclear now. In order to realize the effects of diesel oil on the Zhe oyster, the RAPD (random amplified polymorphic DNA) technique was used. RAPD is a useful assay procedure for the detection of genotoxin-induced DNA damage and mutations. In the present study, the Zhe oysters were exposed to diesel oil at different concentrations and for different exposure periods. The results showed that the DNA band change in RAPD profiles of oysters following diesel oil treatment included loss of normal DNA bands, the appearance of new DNA bands and variations in DNA intensity compared to oysters not exposed to diesel oil. The effects of changes to GTS (genome template stability) were time- and concentration-dependent, the GTS of 10 mg/L was 82.46%, 80.70% and 63.15% in the 8, 16 and 32 days, the GTS of 20 mg/L was 75.44%, 71.93% and 56.14% in the 8, 16 and 32 days, the GTS of 40 mg/L was 73.68%, 70.18% and 56.14% in the 8, 16 and 32 days, respectively. The DNA polymorphisms detected by RAPD analysis could be used as a useful biomarker assay for the detection of genotoxic effects in diesel oil pollution on the oysters, and may be useful for environmental contamination risk assessment.

Genotoxicity potentials of anionic and cationic amino acid-based surfactants

To understand the genotoxic consequences of chemical agents, random amplification of polymorphic DNA (RAPD) as a useful biomarker to be used as an investigation tool for environmental toxicology. In this study, sodium dodecyl sulfate (SDS) was used as a toxic anionic surfactant, and glutamic acid-based cationic bicatanar surfactant (GS) was used as less toxic cationic amino acid-based surfactant. Experimental results show significant correlations between the RAPD profile changes with root growth, mitotic activity and chromosomal aberration test. The inhibitory rates of root growth at 400ppm of SDS and GS were 85% and 32%, respectively. Mitotic activity results showed a drastic decrease in SDS exposures, whereas there was no significant decrease in GS treatment. Comparison of the chromosomal aberration test results, rates were indicated at 100, 200 and 400ppm of SDS and GS; 10, 17, 26 (SDS) and 6, 9, 9 (GS) consequently. Also DNA alterations started at 100 and 200ppm during SDS and GS exposures, respectively. These preliminary findings encourage the utilization of GS as an environmental friendly surfactant detected by these tools in the investigation of genotoxicity potentials of SDS and GS on maize and the other crops.

English

Ornithobacterium rhinotracheale&nbsp;has been identified as a newly emerging respiratory bacterial pathogen that has been causing significant economic losses to the poultry industry. In this study, a total of 30 isolates of&nbsp;O. rhinotracheale&nbsp;isolated&nbsp;(2 from layer pullets, 5 from broilers, 21 from turkeys and 2 standart strains) from chickens and turkeys were characterized by Polymerase Chain Reaction (PCR), RAPD-PCR and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis. The isolates of&nbsp;O. rhinotracheale&nbsp;identified by biochemical tests were also confirmed by PCR. Amplification product of 784 bp was obtained, which is corresponding to the expected size. All of them were characterized as&nbsp;O. rhinotracheale. The&nbsp;random amplified polymorphic DNA&nbsp;(RAPD) profiles using OPG-11 primer and the protein profiles used in SDS-PAGE of the&nbsp;O. rhinotracheale&nbsp;strains show high variability in results of Neighbor Joining (NJ) and Unweighted Pair Group Method with Arithmetic Means (UPGMA). All field strains isolated from turkeys, broilers and layers show different RAPD and protein profiles. &nbsp; Key words:&nbsp;Ornithobacterium rhinotracheale, broiler, layer, turkey, polymerase chain reaction (PCR),&nbsp;random amplified polymorphic DNA-&nbsp;polymerase chain reaction (RAPD-PCR), sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE).