Random Amplified Polymorphic DNA-polymerase Chain Reaction Research Articles

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.

Establishment and characterization of two new cell lines from the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae).

Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

Prolonged Outbreak of Mycobacterium chimaera Infection After Open-Chest Heart Surgery.

Invasive Mycobacterium chimaera infections were diagnosed in 2012 in 2 heart surgery patients on extracorporeal circulation. We launched an outbreak investigation to identify the source and extent of the potential outbreak and to implement preventive measures. We collected water samples from operating theaters, intensive care units, and wards, including air samples from operating theaters. Mycobacterium chimaera strains were characterized by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Case detection was performed based on archived histopathology samples and M. chimaera isolates since 2006, and the patient population at risk was prospectively surveyed. We identified 6 male patients aged between 49 and 64 years with prosthetic valve endocarditis or vascular graft infection due to M. chimaera, which became clinically manifest with a latency of between 1.5 and 3.6 years after surgery. Mycobacterium chimaera was isolated from cardiac tissue specimens, blood cultures, or other biopsy specimens. We were able also to culture M. chimaera from water circuits of heater-cooler units connected to the cardiopulmonary bypass, and air samples collected when the units were in use. RAPD-PCR demonstrated identical patterns among M. chimaera strains from heater-cooler unit water circuits and air samples, and strains in 2 patient clusters. The epidemiological and microbiological features of this prolonged outbreak provided evidence for the airborne transmission of M. chimaera from contaminated heater-cooler unit water tanks to patients during open-heart surgery.

Studying genetic diversity of whitefly B. tabaci Egyptian isolates in relation to some worldwide isolates.

Bemisia tabaci (Gennadius) (Hemiptera, Aleyrodidae) is considered to be one of the most damaging pests in agriculture, causing severe losses in crops worldwide, affecting the tropical and subtropical regions. Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) was used to assess the genetic diversity between different isolates collected from different regions in Egypt compared with some other worldwide isolates of this insect pest. Out of 12 primers 8 primers from Operon technology have shown to differentiate between 13 collected B. tabaci samples from all over Egypt and some other samples collected from different countries with two other populations representing biotypes A and B collected from the US used for biotype demarcation. Using 13 insect samples, RAPD analysis has produced a total number of 72 markers; about 68 polymorphic markers were revealed. The total number of bands obtained for each primer ranged from 4 to 14 within an average of 9 bands per primer. Of the pair wise combination among fifteen populations Ismailia population showed the highest similarity index (0.947), while US biotype A scored the lowest similarity index (0.326). Two major clusters were formed from the UPGMA dendrogram, which was constructed based on Dice similarity coefficient. RAPD-PCR screening demarcated the whitefly population based on the host species and genetic biotypes. Two major clusters have been revealed as A and B with two other minor clusters A1, A2, and B1, B2. Most of the samples collected from Egypt were clustered together in a minor cluster named A1. A1 group is divided into two sub-groups. A1a comprises the populations from Beni-Sweif in Upper Egypt, Ismailia, Kalyobia, El-Fayoum, Tanta, Kafr El-Sheikh, Alexandria, and A1b comprises Spain and Sudan. Group A1a is clustered together based on their host which belongs to the Cucurbitaceae family while Alexandria was separated individually based on its host which is cauliflower. Through the similarity matrix it could be concluded that the populations of Beni-Sweif, Ismailia, Kalyobia, El-Fayoum, Tanta, Kafr El-Sheikh had 80–90% similarity, while the Banha isolate had 30–40% similarity.

Diversity of lactic acid bacteria isolated from Brazilian water buffalo mozzarella cheese.

The water buffalo mozzarella cheese is a typical Italian cheese which has been introduced in the thriving Brazilian market in the last 10 y, with good acceptance by its consumers. Lactic acid bacteria (LAB) play an important role in the technological and sensory quality of mozzarella cheese. In this study, the aim was to evaluate the diversity of the autochthones viable LAB isolated from water buffalo mozzarella cheese under storage. Samples were collected in 3 independent trials in a dairy industry located in the southeast region of Brazil, on the 28th day of storage, at 4 ºC. The LAB were characterized by Gram staining, catalase test, capacity to assimilate citrate, and production of CO2 from glucose. The diversity of LAB was evaluated by RAPD-PCR (randomly amplified polymorphic DNA-polymerase chain reaction), 16S rRNA gene sequencing, and by Vitek 2 system. Twenty LAB strains were isolated and clustered into 12 different clusters, and identified as Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus. Enterococcus species were dominant and citrate-positive. Only the strains of L. mesenteroides subsp. mesenteroides and L. fermentum produced CO2 from glucose and were citrate-positive, while L. casei was only citrate positive. This is the first report which elucidates the LAB diversity involved in Brazilian water buffalo mozzarella cheese. Furthermore, the results show that despite the absence of natural whey cultures as starters in production, the LAB species identified are the ones typically found in mozzarella cheese.

Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

GENETIC DIVERSITY OF OLIVE BARB (Systomus sarana, Hamilton, 1822) FROM DIFFERENT LOCATIONS OF BANGLADESH

Olive barb (Systomus sarana) was collected from three different locations viz. Kuliarchar, Bhairab and Bikrampur in Bangladesh to elucidate genetic diversity by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Six arbitrary oligonucleotide RAPD primers were used to amplify the DNA from each population. A total of 52 bands were produced in 3 olive barb populations where 28 bands were polymorphic indicating 53.84% polymorphisms in those three populations with an average of 9 bands per primer. The molecular size of the amplified DNA fragments ranged between 300 and 2300 bp. 12 unique RAPD bands were observed in the three populations. The bands are specific and stable and thus could be used to characterize each germplasm. The values of pair-wise genetic distances ranged between 0.5232 and 0.8109 with some degrees of genetic variation among the populations. The highest genetic distance (0.8109) was observed between olive barb collected from Kuliarchar and Bikrampur, while the lowest (0.5232) was found between olive barb collected from Kuliarchar and Bhairab. The UPGMA dendrogram segregated three samples of olive barb into two major clusters C1 and C2. The Kuliarchar-Bhairab population pair of olive barb was genetically closer than that of Kuliarchar-Bikrampur. Therefore, RAPD analysis can be used to identify the genetic diversity in olive barb. This information may be used for improved breeding programme and conservation of native olive barb.

Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow's milk cheeses.

“Nostrano-cheeses” are traditional alpine cheeses made from raw cow's milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of “Nostrano-cheeses” and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus.

Identification and characterization of wild lactobacilli and pediococci from spontaneously fermented Mountain Cheese

The Traditional Mountain Malga (TMM) cheese is made from raw cow's milk by spontaneously fermentation in small farms called “Malga” located in Trentino region. This study was designed to characterize the lactic acid bacteria (LAB) growing on MRS medium, of TMM-cheese at the end of the ripening. Ninety-five LAB were isolated and genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. The 95 LAB clustered in 70 biotypes. Pediococcus pentosaceus and Lactobacillus paracasei were the dominant species. Isolates were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase (AP) activity, biogenic amines production, bile salts hydrolysis, conjugated linoleic acid and γ-aminobutyric acid production. Lb. paracasei isolates resulted to be well adapted to Malga environment and to show the best AP activity and acetoin production. TMM-cheese related LAB showed also interesting health promoting properties and produced bioactive substances. In particular, one Lb. brevis biotype produced a GABA mean value of 129 mg/L that is considered a high concentration. The results confirmed that TMM-cheese resident LAB could be exploited for dairy production.

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.

Listeria monocytogenes Serotype Prevalence and Biodiversity in Diverse Food Products

The aim of this study was to assess serotype prevalence and biodiversity of Listeria monocytogenes strains isolated from diverse food products, i.e., minced pork, fruits, and vegetables. Three hundred twenty-six samples previously purchased from supermarkets and street markets within the Athens area were studied for L. monocytogenes prevalence. A total of 121 strains were isolated from the 36 samples that were positive for L. monocytogenes. Serotyping was performed with multiplex PCR, and biodiversity was assessed with random amplified polymorphic DNA (RAPD) PCR analysis using M13, UBC155, and HLWL85 as primers and with repetitive element palindromic (rep) PCR analysis using (GTG)5 as the primer. The majority (17 of 22) of the contaminated minced pork samples contained strains identified as serotype 1/2a, either alone or in combination with strains belonging to serotypes 1/2b, 4a, 4c, or 4ab. However, all L. monocytogenes isolates from fruits and vegetables belonged to serotype 4b. Rep-PCR provided better differentiation of the isolates than did RAPD PCR and resulted in discrimination of the isolates into a larger number of unique profiles. Complete differentiation was achieved only with the combination of these subtyping techniques.

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.

Development of RAPD-SCAR markers for Lonicera japonica (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning.

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with differ- ent species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved.RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR markers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.

Differentiation by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) of Candida albicans isolated from upper respiratory tract in patients with non-small cell lung cancer.

Cancer patients are predisposed to fungal infections caused by Candida albicans, especially to oral or respiratory tract candidiasis. The aim of this study was to estimate genetic diversity by RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) of C. albicans isolated from upper respiratory tract of 100 patients with non-small cell lung cancer. Among 52 strains, 34 genotypes were defined. 10 clusters comprising 28 (53.85%) isolates with similarity coefficient ≥ 80% were formed. The remaining 24 (46.15%) isolates represented individual genotypes. The RAPD-PCR technique revealed genomic variability within C. albicans isolated from upper respiratory tract of the cancer patients.

Investigation of flurbiprofen genotoxicity and cytotoxicity in rat bone marrow cells

This study was performed to investigate cytogenetic effects of NSAID flurbiprofen which was used as active ingredient in some analgesic, antipyretic and anti-inflammatory drugs. Genotoxic effect of flurbiprofen was investigated using in vivo chromosome aberration (CA) test and random amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) test. Also, oxidative stress potential of flurbiprofen was determined by measuring total oxidant and antioxidant level which occurred with flurbiprofen treatment in rat peripheral blood. For these purposes, rats were treated with three concentrations of flurbiprofen (29.25, 58.50 and 117 mg/kg, body weight) in single dose at two different treatment periods (12 and 24 h). According to the results, flurbiprofen did not affect chromosome aberrations in rat bone marrow cells with CA test. In RAPD-PCR test, polymorphic bands were unaffected. Also, test substance did not change total oxidant and antioxidant status (except for 58.50 and 117 mg/kg, 12 h) and therefore it did not lead to significant increase on oxidative stress (again except 58.50 and 117 mg/kg, 12 h). However, flurbiprofen reduced to mitotic indexes and these reductions were dose-dependent for 12 h treatment. In summary, flurbiprofen did not show significant genotoxic effect. But it caused cytotoxicity in rat bone marrow cells.

Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters.

Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

Biomarker System for Genetic Analysis of Stored Grain Pests Callosobruchus maculatus and Callosobruchus chinensis

Random amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used in combination with morphological characters to investigate genetic variations and population structure of Callosobruchus maculatus and Callosobruchus chinensis species from Bundelkhand region of India. It has proved its usefulness to get information quickly in comparatively less-studied species. For RAPD-PCR analysis, out of 25 decanucleotide primers, 15 were selected based on amplification (RAn series). For inter and intra specific analysis, the data indicates that 55 out of total 75 DNA markers detected in both insect pests are polymorphic and can be considered as useful analytical markers. The molecular weight of scorable bands generated by these primers ranges from 150 to ~1500 bp. Mean BS (Band Sharing) value for all primers was scored as 0.228 with 77.2 % mean inter species polymorphism. The highest number of RAPD bands detected for primer RAn 11 was 8 bands while lowest for primer RAn 18 as 2 bands. Out of 15, primer RAn 7, RAn 11, RAn 13 and RAn 18 are considered specific as they produced specific banding pattern for both insect pests in both sexes of different cultures. 100 % sexual and intra species specific genetic similarity was observed. Thus RAPD-PCR technique, along with appropriate statistical procedures, is suitable for the analysis of genetic variations, molecular characterization and relationships among stored grain beetles. The authors have reported the first assessment on the genetic diversity of C. maculatus and C. chinensis populations of central India in the present study.

Assessment of arsenic-induced DNA damage in goldfish by a polymerase chain reaction-based technique using random amplified polymorphic DNA markers.

Arsenic is a groundwater contaminant of global concern. It is a potent human carcinogen, and its marked genotoxic effects have been reported in several human and animal studies. The present work investigates the applicability of the random amplified polymorphic DNA (RAPD) assay to study the DNA-damaging effects of arsenic at low-level exposure in goldfish Carassius auratus. Four experimental groups of fish, A, B, C and D, were exposed to 0, 10, 50, and 1,000 µg L(-1) of arsenic, respectively, in aquaria water for 15 consecutive days. Genomic DNA extraction was followed by RAPD-polymerase chain reaction amplification for each fish separately. One arbitrary decamer primer (PUZ-19) of 33 primers used appeared as the most informative and was capable of exhibiting marked alterations in RAPD profiles between arsenic-exposed and unexposed (control) samples. Different sets of 11 loci were amplified in various experimental groups with four clear polymorphic bands by the primer PUZ-19. The X and XIII amplification loci, which were prominent in the unexposed group, failed to appear in the arsenic-exposed groups. In contrast, the I and XI RAPD bands appeared as new amplification loci in all of the exposed groups. Such alterations in genomic DNA, however, did not exhibit a clear dose-dependent tendency. The RAPD assay, because of its efficacy to unmask alterations in genomic DNA induced by arsenic at low exposure level of 10 µg L(-1), appears to be a sensitive and potential tool for detecting arsenic genotoxicity.

Molecular diagnostic of some nematode species infecting ruminants in Egypt

Molecular techniques are utilized for the diagnosis of parasitic diseases and identification of parasites to enhance the development of specific antigens for serological tests and studying immune response in the patients. In the present study, nematode parasites were Trichuris ovis which were recovered from goats and Trichuris ovis, Haemonchus contortus, and Strongyloides papillosus were recovered from sheep as well Toxocara vitullorum from buffaloes and Toxocara vitullorum from cattle. All samples were checked with 3 different primers by the use of RAPD- PCR and their genetic profiles from different ruminant hosts were compared. Pairwise comparisons were used to evaluate sequence homology and diversity of some variable regions was identified. Interspecific variation in the regions exceeded that within species. Comparison between Toxocara vitullorum from cattle and Toxocara vitullorum from buffaloes revealed genetic polymorphism with genetic variability observed in DNA amplification with primer 3 and non-genetic polymorphism with genetic variability in primer 1 and primer 2 as displayed in figures The use of the Random Amplified Polymorphic DNA - Polymerase chain reaction (RAPD-PCR) technique to amplify short regions of an organism's genome provide more specific method than conventionally employed in epidemiological studies.

English

Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was applied to analyze the genetic variation among Channa striatus collected from four geographically distant locations in India. After initial PCR screening, nine random oligodecamers viz. OPA10, OPA11, OPA15, OPAC01, OPAC03, OPAC05, OPAC07, OPAC09 and OPAC19 which generated the RAPD profile for the four C. striatus populations were selected. Amplification using these nine primers resulted in fragments ranging in length between 309-3029 bp assigned to 87 loci. Estimates of Nei’s (1978) unbiased genetic distance (D) values ranged from 0.3242-0.6320. Unweighted pair group method with arithmetic mean (UPGMA) dendrogram constructed on the basis of genetic distance revealed very close genetic relationship among C. striatus populations of river Tamirabarani (Tamil Nadu) and river Periyar (Kerala). Both populations from river Tamirabarani and Periyar were found to be genetically closer to fish populations from Kolleru Lake (Andhra Pradesh) whereas C. striatus population from Brahmani River (Orissa) was found to be genetically distant from the rest of the populations. Key words: Indian snakehead, Channa striatus, genetic diversity, random amplified polymorphic DNA (RAPD).