Polymorphic DNA Polymerase Chain Reaction Research Articles

Analysis of genetic diversity within and among four rabbit genotypes using biochemical and molecular genetic markers

Genetic variations were detected in four different rabbit genotypes; Animal Production Research Institute (APRI) line, New-Zealand White (NZW), Baladi Black (BB) and Gabali (GAB) breeds. Ten individuals from the first three genotypes and seven individuals from GAB, were blood sampled. Isozymes (esterase and peroxidase) and sodium dodecyl sulphate (SDS)-protein markers were used to detect the genetic variations within these genotypes, whereas the random amplified polymorphic DNApolymerase chain reaction (RAPD-PCR) analysis using six random arbitrary primers were employed to assess the genetic variations and phylogenetic relationships among the four genotypes. The results show a variation in biochemical activity levels since, esterase profiles showed higher percentage of polymorphism (67.21%) than peroxidase (34.78%) and SDS-protein profiles (39.11 %). Moreover, the mean of the genetic similarity within the genotypes based on overall biochemical markers were 0.81 (APRI), 0.91 (NZW), 0.89 (BB) and 0.86 (GAB), which indicates high homogeneity within each of the four genotypes. The polymorphism percentage based on overall RAPD primers was 35.44% among these genotypes, which presents a kind of genetic diversity. From the dendrogram tree, the genotypes BB and GAB appeared to be closely related, while the APRI genotype was the most different. Keywords : Rabbits, genetic diversity, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE), isozymes, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) African Journal of Biotechnology Vol. 12(20), pp. 2830-2839

Distribution and epidemiology of genotypes of the crayfish plague agent Aphanomyces astaci from noble crayfish Astacus astacus in Finland

The crayfish plague agent Aphanomyces astaci was isolated from 69 noble crayfish Astacus astacus samples in Finland between 1996 and 2006. All isolates were genotyped using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Altogether, 43 isolates belonged to the genotype group of Astacus strains (As), which is assumed to represent the genotype originally introduced into Europe around 1860 and into Finland in 1893. There were 26 crayfish plague isolates belonging to the group of Pacifastacus strain I (Ps1), which appeared in Europe after the stocking of the North American species signal crayfish Pacifastacus leniusculus. The geographical distribution of the 2 genotypes in Finland corresponded with the stocking strategies of signal crayfish. The majority of Ps1-strains (83%) were associated with a classical crayfish plague episode involving acute mortality, compared with only 33% of the As-strains. As-strains were found more often by searching for reasons for population declines or permanently weak populations, or through cage experiments in connection with reintroduction programmes. In some water bodies, isolations of the As-strains were made in successive years. This study shows that persistent crayfish plague infection is not uncommon in noble crayfish populations. The described epidemiological features suggest a difference in virulence between these 2 genotypes.

Large outbreak of cholera caused by El Tor variant Vibrio cholerae O1 in the eastern coast of Odisha, India during 2009.

A large outbreak of cholera reported during April-July 2009 in the Kendrapada district of Odisha, India was investigated. Forty-one rectal swabs and 41 water samples, collected from diarrhoeal patients and from different villages were bacteriologically analysed for the isolation of bacterial enteriopathogens, antibiogram profile and detection of various toxic genes. The bacteriological analysis of rectal swabs and environmental water samples revealed the presence of V. cholerae O1 Ogawa biotype El Tor. The V. cholerae strains were resistant to ciprofloxacin, co-trimoxazole, chloramphenicol, streptomycin, ampicillin, furazolidone and nalidixic acid. The multiplex polymerase chain reaction (PCR) assay on V. cholerae strains revealed the presence of ctxA and tcpA genes. The mismatch amplification of mutation assay (MAMA) PCR on clinical and environmental isolates of V. cholerae revealed that the strains were El Tor biotype, which harboured the ctxB gene of the classical strain. The random amplified polymorphic DNA PCR analysis and pulsed-field gel electrophoresis results indicated that the V. cholerae isolates belonged to the same clone. This investigation gives a warning that the El Tor variant of V. cholerae has spread to the coastal district causing a large outbreak that requires close monitoring and surveillance on diarrhoeal outbreaks in Odisha.

Effects of garlic on Schistosoma mansoni harbored in albino mice: Molecular characterization of the host and parasite

Garlic has been used for its health benefits for thousands of years. Modern research confirmed many of the healing properties of garlic, including its antiparasitic activity. This study was designed to evaluate the antischistosomal action of garlic through detecting the changes in DNA profile of Schistosoma mansoni worms and the infected mouse. Forty mice were subcutaneously infected with ~200 Schistosoma mansoni cercariae/mouse. Infected mice were divided into four equal groups: non-treated, prophylactic, therapeutic, and continuously-treated. Non-infected control and garlic-treated groups were assigned for the sake of comparison. Garlic extract (50mg/kg bw/mouse) was given orally, day after day, at a fixed daytime. Seven weeks post-infection, adult schistosomes were recovered by perfusion and the livers of the mice were excised out and were processed for DNA extraction and Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The results showed that garlic exerted no major changes in the genome of schistosomes. Nevertheless, that schistosomal infection induced genetic alterations in the DNA of mice, and garlic was able to ameliorate such alterations to a great extent.

RAPD-PCR and real-time Pcr Hrm based genetic variation evaluations of <i>Urtica dioica</i> parts, ecotypes and evaluations of morphotypes in Turkey

Urtica dioica is an ethnobotanically and medicinally important Complementary and Alternative Medicine (CAM) plant worldwide and in Turkey; 90 % of herbal CAM applications depend on it in Turkey. It has a wide range of habitats in nearly all continents. It is found in all three phytogeographical regions in Turkey (Euro-Siberian, Irano-Turanian, Mediterranean) with high adaptivity to heterogeneous geographies such as climate, soil types and altitudes. This fact in relation to the assessment of chemical constituents of the plant and combining with further genetic and morphological variation data can assist and enhance the works for the utility and reliability of CAM applications in effect and activity of this plant species. In this work we have made some preliminary experiments with novel approaches to reveal the ecotypes and genetic variation of mighty ecotypes of Urtica dioica from different phytogeographical regions of Turkey (Euro-Siberian and Mediterranean). The ecotypes have heterogeneity in both its parts (leaf, stem, root) as revealed by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) using random primers and High-resolution Melt (HRM) analysis using Urtica dioica specific primers and universal chloroplast DNA (cpDNA) primers and morphological traits such as phenolic contents and antioxidant capacities of plants' leaf infusions as used in medicinal applications in Turkey. This work will contribute a lot for the development of molecular markers to detect the genetic variation and heterogeneity of Urtica dioica to further relate with expected phenotypes that are most useful and relevant in CAM applications.

Detection of Carbapenemase-Encoding Genes Among Clinical Isolates of Pseudomonas aeruginosa in a Chinese Burn Unit

The carbapenemases have recently emerged as molecules implicated in one of the most feared bacterial resistance mechanisms because of their ability to hydrolyze virtually all lactamase agents and their highly mobile genes. This study aimed to investigate the prevalence of carbapenemase and antimicrobial susceptibilities of Pseudomonas aeruginosa isolated from burn patients in Chongqing, China. Antimicrobial susceptibility of 111 isolates was determined by the disc agar diffusion test and the agar dilution method. Random Amplification of Polymorphic DNA polymerase chain reaction analysis revealed 111 P. aeruginosa 42 genotypes. Carbapenemase genes were amplified by polymerase chain reaction and the sequence verified by blast. Ninety-three of 111 (83.8%) isolates were resistant to imipenem; all of them had developed multidrug resistance and exhibited higher resistant rates compared with the imipenem-susceptible Pseudomonas. Ciprofloxacin was the most effective antipseudomonal agent. Thirty-three of the isolates were identified to contain the metallo-β-lactamase blaIMP-4 gene and belong to different Random Amplification of Polymorphic DNA polymerase chain reactiongenotypes. In conclusion, the high prevalence of multidrug resistance (94.6%) and the production of blaIMP-4 genes in P. aeruginosa isolates in burn patients highlight the necessity of considering these issues in burn hospitals.

MOLECULAR CHARACTERIZATION OF GOATS USING RANDOM AMPLIFIED POLYMORPHIC DNA

Black Bengal goat is a very valuable goat breed in India. The present investigation was proposed to be undertaken to evaluate gene flow, phylogeographic history, genetic structure and differentiation of both the goat populations to study genetic variation prevail ing among Black Bengal and Black Bengal type goat in west Bengal and Jharkhand respectively and to study genetic relationship between both goat populations and to study population structure and gene flow between th ese two goat populations. This experiment was conducted in the department of animal breeding and genetics ( molecular biology laboratory). DNA was extracted from 50 goats from each of the Black Bengal and Black Bengal type goats of Jharkhand and the extracted DNA was observed by gel electrophoresis. Ten specific goat specific primers were obtained from sigma. All the primers except for primers one and two were capable of priming polymorphic amplification pattern in both the b reeds. Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis was carried out using DNA samples of 50 Black Bengal goat and 50 Black Bengal type goats of Jharkhand (referred to as Jharkhand black) goat breeds. Only unambiguous, reproducible and scorable polymorphic fragments were taken into consideration for analysis. The amplific ation pattern of representative samples of Black Be ngal showed that primers 6,7,8,9 were highly polymorphic. Polymorphism Information Content (PIC) or expected heterozygosity scores varied from a minimum of 0.219 for primer sigma 06-3 to a maximum of 0.486 for primer sigma 04 with over all mean 0.411 in Black Bengal goat. Similarly for Jharkhand Black, the valu e of PIC ranged from a minimum of 0. 278 for primer sigma 06-3-0.496 for primer sigma10-2 with over all mea n value of 0.413. Data were analyzed by using a computer programe POPGENE (Version 1.31). A more appropriate measure of genetic variation within a p opulation is gene diversity .It was observed that, mean value of Nei,s Gene diversity value (h) was 0.3750 for Black Bengal and was 0.4022 for Jharkhand black. In case of Black Bengal, it was found that the maximum value of Nei’s gene diversity was 0.4861 for locus sigma 6-2 and sigma 9-2 while the minimum value of 0.0664 was found for locus Sigma 04. In Jharkhand Black t he maximum value of Nei’s gene diversity (h) was found to be 0.4965 for locus Sigma 10-2 and sigma06-1and the minimum value was 0.2491 for locus sigma 08-1. Dendrogram based on Nei’s genetic distance using Unweighted Pair Group Method of Arithmetic Means (UPGMA) indicated relative closeness and distance of the two goat breed. This study was mainly done on b reeds raised in marginal agricultural areas in orde r to assist in situ conservation. The result was importa nt with respect to puzzle of goat genetic diversity and conservation. Therefore it can be recommended that within breed diversity is actively maintained to en able these extensively unmanaged stocks to adapt to futu re demands and conditions and there is ample scope for further improvement in its productivity through app ropriate breeding strategies.

Genomic DNA Extraction from Ferula jaeschkeana Vatke (Apiaceae) Optimized for Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) analysis

A protocol for rapid extraction of high quantity and quality of genomic DNA and Random Amplified Polymorphic DNA Polymerase Chain Reaction (PCR) analysis has been optimized for Ferula jaeschkeana Vatke (Apiaceae), an overexploited medicinally important species of Western Himalaya in India. The protocol utilizes preserved leaf material for DNA extraction. Liquid nitrogen was used for grinding the deveined leaf tissue. The isolated DNA was essentially free of polyphenolics, polysaccharides and other major contaminants as revealed by color difference of DNA isolated by the available protocols and the present one. Quantification of the isolated DNA was performed in a UV spectrophotometer (UV1800, Shimadzu) and the purity determined by calculating the ratio of absorbance at 260 nm to that of 280 nm (260/280 = 1.8–2.0). The quality and quantity of DNA was also visualized on 0.8 % agarose gel by comparing band intensity with that of standard amount of lambda DNA (165 μg/ml, Banglore Genei Pvt. Ltd., India). The isolated DNA was diluted to 25 ng/μl which was used for PCR amplification.

Importance of on-farm management practices on lactate-fermenting Clostridium spp. spore contamination of Manchega ewe milk: Determination of risk factors and characterization of Clostridium population

This work studies the on-farm management practices that increase the risk of developing high lactate-fermenting Clostridium spp. spore counts in ewe milk. Moreover the ecology of the Clostridium population was studied in relation to those management practices. A total of 136 bulk tank milk samples belonging to 23 Manchega ewe flocks were analysed for lactate-fermenting Clostridium spores by the most probable number technique. Information about the housing conditions, the feeding characteristics and the milking parlour hygiene were also collected from the 23 flocks. A logistic regression analysis indicated the farm-made total mixed ration, the wet brewers’ grains used for feeding, and the presence of dust in the milking parlour as the on-farm management risk factors that lead to an increase of Clostridium spp. spore counts. A total of 114 Clostridium isolates from milk samples were typed using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The 35 different genotypes obtained were subsequently identified by restriction analysis of 16S-rRNA gene (16S-ARDRA), so three populations were observed: 93.86%, 3.51% and 2.63% corresponding to Clostridium sporogenes, Clostridium beijerinkii and Clostridium tyrobutyricum respectively. The risk factors of high Clostridium spp. spore counts also increased Clostridium population diversity and favoured the presence of C. beijerinkii and C. tyrobutyricum. Moreover, genotypes of these species were the most gas-productive ones. The results confirmed that these risk factors should be taken into account in developing strategies in the control of Clostridium spp. spores contamination in ewe milk.

Molecular typing of the fresh water snail Lymnaea arabica, the possible intermediate host of Fasciola hepatica, collected from Saudi Arabia, by RAPD-PCR

Fasciolosis is caused by Fasciola hepatica with Lymnaea spp. as intermediate hosts. Lymnaea arabica, as a possible snail host collected from the Kingdom of Saudi Arabia, was investigated by Random Amplified Polymorphic DNA polymerase chain reaction (RAPD-PCR) technique. Polymorphic bands from L. arabica were sought for fingerprinting (typing) and identification of this species. The resultant electrophoretic patterns would be used as molecular bio-markers for thesemolluscas. This will help in the identification of the snails at a molecular level for the first time in the country that, in turn, will help in disease control. This technique was proved to be an accurate, appropriate, efficient and valuable alternative to traditional identification tools of Lymnea spp.

English

Sheep is an important livestock species of Tunisia. They contribute greatly to the food safety of the country and in the livelihood of a large number of small and marginal farmers and landless labourers engaged in sheep rearing. In this study, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis was used to assess the genetic difference and gene flow among two Tunisian sheep breeds (the Barbarine and the Western thin tail). A total of 62 bands were detected with an average of 7.75 bands per primer. The unweighted pair-group method with arithmetic average (UPGMA) and principal component analysis (PCA) showed a clear differentiation between the two studied breeds. Genetic differentiation coefficient (Gst) over all loci was 0.1922, the fixation index [Fst by Analysis of molecular variance (AMOVA)] was 0.308 (P<0.001), and the gene flow value (Nm) was 1.3102. It is clear from this study that Barbarine and Western thin tail breeds are completely distinct and that they show high and significant genetic differentiation. However, the level of gene migration between them is quite high and may lead eventually to the loss of breed’s purity.  Key words: Sheep, genetic differentiation, gene flow, RAPD-PCR

English

  Mobile phones are increasingly used by professionals, university staffs and health care personnel for communication. These can harbor various potential pathogens. This study evaluates and identifies the bacterial contamination rate of mobile phones in the university setting that are in frequent contact with faculty members, personnel, students and/or physicians and nurses in the university clinic. A total of 101 mobile phones belonging to different categories working in various departments at Taif University, KSA were screened for microorganisms’ contamination. Out of the total 101 mobile phones, growth was obtained in 78 (77.2%) mobile phones; 70 (89.7%) from staffs, personnel, students and 8 (10.3%) from clinical workers. Staphylococcusspp and Bacillus spp were the most commonly isolated organisms. Coagulase negative Staphylococcus was the most frequently isolated; 60 (27.12%). The efficacy of decontamination with 70% isopropyl alcohol was found to be 71.3%, as only 29 mobile phones showed growth after decontamination. It was found that around 61.5% of the mobile phones of health care workers at university clinic were contaminated and thus acted as a potential source of nosocomial infections. According to morphological, physiological characteristics, APi profiles and sequencing of 16S-rRNA gene, the selected eight isolates were identified as Bacillus pumilus, Bacillus cereus,Staphylococcus aureus, Staphylococcus hominis, Staphylococcus succinus,Staphylococcus xylosus and Staphylococcus saprophyticus. Based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), 32 unique RAPD fragments were identified among the selected isolates. Such unique fragments could be considered as specific markers and might be utilized in tracking the bacterial isolates.   Key words: Mobile phones, contamination, pathogen carriers, coagulase negative staphylococci, Bacillus species, 16S-rRNA, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR)

Genetic diversity analysis of Bemisia tabaci populations in Pakistan using RAPD markers

Background: The Bemisia tabaci is one of the most devastating pests of agricultural crops and ornamental plants worldwide. The genetic diversity and biotype status of the Bemisia tabaci in Pakistan was assessed by using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). A total 80 samples of B. tabaci collected from 14 districts of the Punjab province and 7 districts of the Sindh province were included. Results: All 10 primers screened in this study generated 151 scorable amplification products, of which 117 or 77% were polymorphic. Pairwise Nei and Li’s similarity had ranged from 0.25 to 0.88 among all individuals analyzed. Based on Nei and Li’s similarity coefficients Bemisia populations were grouped into 3 main clusters and clearly distinguished the non B biotype from the B biotype. Conclusion: The level of similarity among populations of same biotypes was high whereas between populations of non B and B biotypes appeared to be less closely related. This analysis showed that non B biotype is prevalent in both provinces however B biotype is restricted to few locations in Sindh. This monitoring of the spread of B. tabaci in Pakistan will assist in the establishment of appropriate management strategies.

English

Molecular methods have been established to make better sensitivity and specificity for differentiation of pathogenic and allergenic fungi. The purpose of this study was to obtain an appropriate molecular pattern for differentiation of Penicillium species understudy. In this study, 30 isolates of four Penicillium species including Penicillium citrinum,Penicillium notatum, Penicillium oxalicum and Penicillium frequentans were used. First, isolates were cultured. Then, for obtaining the fungal mats, fungi were grown into Sabouraud glucose broth. Freeze and Thaw, and glass beads were used for cell disruption, and DNA was extracted by phenol-chloroform method. RAPD-PCR technique was performed using three primers. The results showed that each primer produced a different reproduction pattern. A total of 2 to 17 bands were observed in all the isolates ofPenicillium, except P. citrinum C2 and P. frequentans F7. According to primer 1, molecular patterns of Penicillium species were approximately similar, but multiplied patterns were not completely similar. Having used primer 2, multiplied patterns in P. oxalicum and P. notatum were to some extent similar, but there were some observed differences between P. frequentans and two other species P. oxalicum and P. notatum.P. citrinum, P. oxalicum and P. notatum had approximately the same patterns using primer 3, but some differences were observed in P. frequentans with three other species.In conclusion, the interspecies differences were demonstrated with all the primers. Primers 1 and 2 were able, to some extent, to differentiate these species, while primers 1 and 3 separated P. frequentans from the others.   Key words: Penicillium, molecular pattern, random amplified polymorphic DNA-polymerase chain reaction, molecular bands.

Isolation of a UDP-glucose: Flavonoid 5-O-glucosyltransferase gene and expression analysis of anthocyanin biosynthetic genes in herbaceous peony (Paeonia lactiflora Pall.)

Herbaceous peony (Paeonia lactiflora Pall.) is an excellent material for studying the formation of flower colour because of its abundant colour. The full-length cDNA of a UDP-glucose: Flavonoid 5-O-glucosyltransferase gene (UF5GT) containing 1629 bp nucleotides was obtained from P. lactiflora. The expression patterns of nine related anthocyanin biosynthetic genes (PlPSY, PlCHS, PlCHI, PlF3H, PlF3'H, PlDFR, PlANS, PlUF3GT and PlUF5GT) in diurnal variation petals showed that their expression peaks were basically at 15:00 and the expression patterns were consistent with the trend of sampling conditions except individual gene. And the highest expression levels were in PlCHS, PlDFR and PlUF3GT, which could be the candidates to regulate P. lactiflora flower colour by means of genetic engineering.

Study on morphology, pathogenicity and genetic diversity of Wilsonomyces carpophilus isolates, the causal agent of shot hole of stone fruit trees based on RAPD-PCR in Iran

Shot hole disease is one of the most important diseases of stone fruit trees in Iran. The disease is wide spread among orchards of Prunus spp. During spring and summer of 2007, 80 monoconidial isolates of the pathogen were recovered from infected leaves, fruits and twigs of different Prunus spp. in West Azerbaijan, Tehran, Ghazvin and Razavi Khorasan provinces of Iran and were studied taxonomically. Based on morphological and physiological characteristics and growth optimal temperature, all isolates were identified as Wilsonomyces carpophilus. Seedlings of stone fruits (apricot, almond, peach, nectarine, plum, sweet cherry and sour cherry) were used for pathogenicity tests. All seedlings were susceptible to the fungal isolates and showed disease symptoms on twigs, leaves, buds and petioles. Genetic diversity of 28 selected fungal isolates was investigated based on DNA fingerprinting by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), using four random primers. Based on cluster analysis of the PCR results from the four primers, 10 fingerprinting groups (clonal lineages) and 27 haplotypes were identified. Clonal lineages “C”, “D” and “E”, each with six haplotypes formed the biggest clonal lineages, but other clonal lineages (“B”, “F”, “G”, “H”, “I” and “J”) included only one isolate. No correlation was detected among clonal lineages with the location of selected isolates and their host species. A correlation was found between the substrate (fruit, twig or leaf) and clonal lineages, particularly in “C” clonal lineage. The results showed that the fungus population had high genetic diversity which is distributed among the different areas of Iran.

Coexistence of armA and genes encoding aminoglycoside-modifying enzymes in Acinetobacter baumannii

A total number of 104 Acinetobacter baumannii isolates obtained from a teaching hospital were studied for the genetic basis of their resistance to aminoglycosides. The rate of aminoglycoside-resistant A. baumannii was 76.9% (80/104), and 66.25% (53/80) aminoglycoside-resistant isolates were high-level resistant to either aminoglycosides including amikacin, gentamicin and tobramycin (MIC≥512μg/ml). Genes encoding aminoglycoside-modifying enzymes including ant(2″)-Ia, aac(6′)-Ib, aph(3′)-Ia, aac(3)-Iaand aph(3′)-VI, and 16S ribosomal RNA (rRNA) methylase armA, rmtA, rmtB, rmtC, rmtD,rmtE and npmA were detected by polymerase chain reaction (PCR), armA gene waspresent in 50% (52/104) of A. baumannii isolates with high-level resistance to aminoglycosides (MICs≥512 μg/ml). No rmtA, rmtB, rmtC, rmtD, rmtE or npmA genes were found. The rates of ant(2″)-Ia, aac(6′)-Ib, aph(3′)-Ia, aac(3)-Ia and aph(3′)-VI were 68.25%,20%,70%,47.5% and 1.25%, respectively. armA, ant(2″)-Ia and aph(3')-Ia genes coexisted in 62.5%(50/80) of aminoglycoside-resistant isolates, armA, ant(2″)-Ia and aac(6′)-Ib were commonly positive in same isolates. The presence of aminoglycoside-modifying enzymes was responsible for the moderate level resistance to aminoglycosides in A. baumannii, whereas armA was responsible for the high-level resistance to aminoglycosides. Aminoglycoside-resistant A. baumannii isolates were classified into genotypes by Random Amplified Polymorphic DNA PCR (RAPD-PCR), pattern A was almost throughout wards of the hospital. In summary, most A. baumannii is resistant to aminoglycosides, in the hospital coexistence of armA and genes encoding aminoglycoside-modifying enzymes are the main reasons. Key words: Acinetobacter baumannii, aminoglycoside-resistant, 16S rRNA methylase, aminoglycoside-modifying enzymes, RAPD-PCR

Toxicity of Bacillus thuringiensis d-exotoxins and b-endotoxins to Drosophila melanogaster, Ephestia kuhniella and human erythrocytes

A total of 73 Bacillus thuringiensis (Bt) strains were screened for the presence of non-hemolytic insecticidal β -exotoxin-free δ -endotoxins. Out of them, 45 Bt strains produced δ -endotoxins with specific insecticidal activity against Drosophila melanogaster and/or Ephestia kuhniella larvae. The thermostable β -exotoxin was observed only in 15 Bt strains and appeared to exhibit dual non-specific insecticidal activity against both D. melanogaster and E. kuhniella larvae and showed in vitro hemolysis for human erythrocytes. It was found that β -exotoxin was produced by Bt strains belonging to five serovars ( israelensis , kenyae , kurstaki , pakistani , and tohokuensis ) and two non-serotypable strains. This result suggests that β-exotoxin production is a strain-specific property rather than a serovar-specific property. To our knowledge, this is the first study that demonstrates β -exotoxins production association with Bt strains belonging to serovars israelensis, pakistani, and tohokuensis. The plasmid DNA profiles of some β -exotoxin producing Bt strains shared large plasmid patterns which may have the common β -exotoxin regulatory gene(s). It was found that 16 local Bt strains, 15 of which belonged to five serovars ( aizawai , israelensis , kurstaki , morrisoni , and pakistani ) and one was autoagglutinated strain, produced non-hemolytic insecticidal β -exotoxin-free δ -endotoxins. Based on random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), the genotypic relatedness among these 16 Bt strains was investigated. The strains were grouped into two clusters. Bt strains within serovars israelensis were grouped in two subclusters, Bt strains within serovars aizawai were genomically homogeneous and clustered together, while the other serovars were grouped together in one subcluster. The autoagglutinated strain was clustered within serovar israelensis . Thus, these δ -endotoxins can be developed for the use in Bt-based insecticidal preparations. Keywords: Endotoxin, exotoxin, hemolytic, serovar, thuringiensis

Preliminary molecular analysis of the genetic diversity of some Atriplex species present in the Northeast of Algerian

In the arid and semi arid areas, salt bush ( Atriplex ) represents an important forage resource. The characterization of the genetic diversity of these species is useful for their classification, their conservation and their improvement. In this context, we used the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique to test the genotypes of three different species of Atriplex: Atriplex halimus, Atriplex canescens and Atriplex nummularia . The Obtained results showed 319 amplified bands which were mostly polymorphic. The analysis of the results generated four different groups where the genotypes of two introduced species (A. canescens and A. nummularia) were clustered into two independent groups. However, genotypes of the autochthonous species ( A. halimus ) was subdivided into two different independent groups where we noted that individuals of one of these groups are genetically the nearest between them, by report, to the existing others within the same group. In general, results reveal inter and intra high level of genetic diversity in the Atriplex studied species. Key words: Atriplex, Chenopodiaceae, genetic diversity, molecular analysis, steppe, Algeria.

Selection of Dominant NSLAB from a Mature Traditional Cheese According to their Technological Properties and in vitro Intestinal Challenges

Isolates (47) of lactobacilli from 5 different productions of Melichloro cheese were examined for potential use as adjunct cultures. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins classified 29 isolates as L. paraplantarum and 18 as L. paracasei subsp. paracasei. Randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis differentiated the L. paraplantarum and L. paracasei subsp. paracasei isolates at strain level and both, RAPD analysis and whole-cell protein profiling provided useful information about the diversity of nonstarter lactic acid bacteria (NSLAB) in the different cheese productions. The isolates were slow acidifiers and about 70% of them degraded, preferentially α(s)-casein. The amounts of amino acids accumulated in the milk increased with the incubation time. A similar enzyme profile was exhibited by strains of both species, except for α-mannosidase and α-fucosidase, which were not detected in the L. paracasei subsp. paracasei strains. All strains grew in the presence of bile at 0.3% and the majority was able to withstand pH 2.5 and pancreatin at 0.1%. Moreover, all strains reduced cholesterol in vitro, with higher removal ability recorded for strains of L. paraplantarum. A narrow spectrum of antibacterial activity was recorded for 88% of the strains. Selected isolates with appropriate technological and interesting in vitro intestinal challenges could be used as adjuncts and deserve further studies.