Search results for: ISSR primers

Authors: Nuha Mansour Alhazmi, Magda Mohamed Aly, Fardus M. Bokhari, Ahmed Bahieldin, Sherif Edris

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Out of 30 fungal isolates, 2 new isolates were proven to degrade poly-β-hydroxybutyrate (PHB). Enzyme assay for these isolates indicated the optimal environmental conditions required for depolymerase enzyme to induce the highest level of biopolymer degradation. The two isolates were basically characterized at the morphological level as Trichoderma asperellum (isolate S1), and Aspergillus fumigates (isolate S2) using standard approaches. The aim of the present study was to characterize these two isolates at the molecular level based on the highly diverged rRNA gene(s). Within this gene, two domains of the ribosome large subunit (LSU) namely internal transcribed spacer (ITS) and 26S were utilized in the analysis. The first domain comprises the ITS1/5.8S/ITS2 regions ( > 500 bp), while the second domain comprises the D1/D2/D3 regions ( > 1200 bp). Sanger sequencing was conducted at Macrogen (Inc.) for the two isolates using primers ITS1/ITS4 for the first domain, while primers LROR/LR7 for the second domain. Sizes of the first domain ranged between 594-602 bp for S1 isolate and 581-594 bp for S2 isolate, while those of the second domain ranged between 1228-1238 bp for S1 isolate and 1156-1291 for S2 isolate. BLAST analysis indicated 99% identities of the first domain of S1 isolate with T. asperellum isolates XP22 (ID: KX664456.1), CTCCSJ-G-HB40564 (ID: KY750349.1), CTCCSJ-F-ZY40590 (ID: KY750362.1) and TV (ID: KU341015.1). BLAST of the first domain of S2 isolate indicated 100% identities with A. fumigatus isolate YNCA0338 (ID: KP068684.1) and strain MEF-Cr-6 (ID: KU597198.1), while 99% identities with A. fumigatus isolate CCA101 (ID: KT877346.1) and strain CD1621 (ID: JX092088.1). Large numbers of other T. asperellum and A. fumigatus isolates and strains showed high level of identities with S1 and S2 isolates, respectively, based on the diversity of the first domain. BLAST of the second domain of S1 isolate indicated 99 and 100% identities with only two strains of T. asperellum namely TR 3 (ID: HM466685.1) and G (ID: KF723005.1), respectively. However, other T. species (ex., atroviride, hamatum, deliquescens, harzianum, etc.) also showed high level of identities. BLAST of the second domain of S2 isolate indicated 100% identities with A. fumigatus isolate YNCA0338 (ID: KP068684.1) and strain MEF-Cr-6 (ID: KU597198.1), while 99% identities with A. fumigatus isolate CCA101 (ID: KT877346.1) and strain CD1621 (ID: JX092088.1). Large numbers of other A. fumigatus isolates and strains showed high level of identities with S2 isolate. Overall, the results of molecular characterization based on rRNA diversity for the two isolates of T. asperellum and A. fumigatus matched those obtained by morphological characterization. In addition, ITS domain proved to be more sensitive than 26S domain in diversity profiling of fungi at the species level.

Keywords: Aspergillus fumigates, Trichoderma asperellum, PHB, degradation, BLAST, ITS, 26S, rRNA

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Authors: Siwapech Silapaprayoon, Januluk Khanobdee, Sompid Samipak

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Chili peppers are the fruits of Capsicum pepper plants well known for their fiery burning sensation on the tongue after consumption. They are members of the Solanaceae or common nightshade family along with potato, tomato and eggplant. Thai cuisine has gained popularity for its distinct flavors due to usages of various spices and its heat from the addition of chili pepper. Though being used in little quantity for each dish, chili pepper holds a special place in Thai cuisine. There are many varieties of chili peppers in Thailand, and thirty accessions were collected at Rajamangala University of Technology Lanna, Lampang, Thailand. To effectively manage any germplasm it is essential to know the diversity and relationships among members. Thirty-six Inter Simple Sequence Repeat (ISSRs) DNA markers were used to analyze the germplasm. Total of 335 polymorphic bands was obtained giving the average of 9.3 alleles per marker. Unweighted pair-group mean arithmetic method (UPGMA) clustering of data using NTSYS-pc software indicated that the accessions showed varied levels of genetic similarity ranging from 0.57-1.00 similarity coefficient index indicating significant levels of variation. At SM coefficient of 0.81, the germplasm was separated into four groups. Phenotypic variation was discussed in context of phylogenetic tree clustering.

Keywords: diversity, germplasm, Chili pepper, ISSR

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Authors: Vivek Vaishnav, Shamim Akhtar Ansari

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Teak (Tectona grandis L.f.) belonging to plant family Lamiaceae and the most commercialized timber species is endemic to South-Asia. The adaptive fitness of the species metapopulation was evaluated through its genetic differentiation and assessing the influence of geo-climatic conditions. 290 genotypes were sampled from 29 locations of its natural distribution and the genetic data was incorporated with geo-climatic parameters. Through Bayesian approach based analysis of 43 highly polymorphic ISSR markers, six homogeneous clusters (0.8% genetic variability) were identified. The six clusters were found with the various regimes of the temperature range, i.e., I - 9.10±1.35⁰C, II -6.35±0.21⁰C, III -12.21±0.43⁰C, IV - 10.8±1.06⁰C, V - 11.67±3.04⁰C, and VI - 12.35±0.21⁰C. The population had a very high percentage of LD (21.48%) among the amplified loci possibly due to experiencing restricted gene flow as well as co-adaptation and association of distant/diverse loci/alleles as a result of the stabilized climatic conditions and countless cycles of historical recombination events on a large geological timescale. The same possibly accounts for the narrow distribution of teak as a climax species in the tropical deciduous forests of the country. The regions of strong LD in teak genome significantly associated with climatic parameters also reflect that the species is tolerant to the wide regimes of the temperature range and may possibly withstand global warming and climate change in the coming millennium.

Keywords: Bayesian analysis, inter simple sequence repeat, linkage disequilibrium, marker-geoclimatic association

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Authors: Fitri Rahmi Fadhilah, Edhyana Sahiratmadja, Ani Melani Maskoen, Ratu Safitri, Supartini Syarif, Herman Susanto

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Cervical cancer is the second most common cancer in women after breast cancer. In Indonesia, the incidence of cervical cancer cases is estimated at 25-40 per 100,000 women per year. Human papillomavirus (HPV) infection is a major cause of cervical cancer, and HPV-16 is the most common genotype that infects the cervical tissue. The major late protein L1 may be associated with infectivity and pathogenicity and its variation can be used to classify HPV isolates. The aim of this study was to determine the phylogenetic tree of HPV 16 L1 gene from cervical cancer patient isolates in Bandung. After confirming HPV-16 by Linear Array Genotyping Test, L1 gene was amplified using specific primers and subject for sequencing. Phylogenetic analysis revealed that HPV 16 from Bandung was in the subgroup of Asia and East Asia, showing the close host-agent relationship among the Asian type.

Keywords: L1 HPV 16, cervical cancer, bandung, phylogenetic

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Authors: Cherity A. Ysquierdo, Pia U. Olafson, Donald B. Thomas

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Musca domestica L. were collected from cattle diagnosed with bovine ringworm to evaluate the potential of the house fly to disseminate Trichophyton verrucosum E. Bodin, a fungal dermatophyte that is the causative agent for ringworm in cattle. Fungal isolates were cultured from 45 individual flies on supplemented Sabouraud dextrose agar, and isolates were identified using morphological and microscopic approaches. Each isolate was further identified by PCR amplification of the ribosomal DNA locus with fungal specific primers and subsequent amplicon sequencing. No T. verrucosum were identified using these approaches. However, 36 different fungal species representing 17 genera were cultured from these flies, including several allergenic and pathogenic species. Several species within the fungal orders Hypocreales, Microascales, Onygenales, Saccharomycetales, Xylaniales, and Agaricales were observed for the first time on house flies. The most frequent fungus recovered was Cladosporium cladosporoides, which is known to be a ubiquitous, airborne allergen.

Keywords: bovine ringworm, Cladosporium, dermatophyte, Musca domestica

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Authors: M. Khallaf, R. Khalil, H. Ghetas

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In the present study, V. harveyi and V. alginolyticus were isolated from cultured seabass and seabream at Damietta Governorate, Egypt, during summer season. Isolates were biochemically and molecularly identified using primers for Vhh and Collagenase genes. The most prominent clinical observations of diseased fish were exophthalmia, abdominal distension, and multifocal cutaneous hemorrhagic ulceration on the dorsal musculature and caudal peduncle. Physicochemical characteristics of water samples indicated that the unionized ammonia, nitrate, and hydrogen sulphate concentrations were higher than the acceptable limits. Heavy metals concentrations in water samples exhibited higher concentrations than the permissible levels for fish culture, which was considered as chemical stressors that increase the prevalence of these bacterial diseases. Immune parameters were lower in diseased seabass and seabream than apparently healthy fish. Lesions of different fish organs were identified histopathologically.

Keywords: seabass, seabream, Vibrio alginolyticus, Vibrio harveyi

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Authors: Taru Singh, Shukla Das, V. G. Ramachandran

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Objectives: To develop SYBR green based real-time PCR assay to detect carbapenemases (NDM, IMP) genes in E. coli. Methods: A total of 40 E. coli from stool samples were tested. Six were previously characterized as resistant to carbapenems and documented by PCR. The remaining 34 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial RNA was extracted using manual method. The real-time PCR was performed using the Light Cycler III 480 instrument (Roche) and specific primers for each carbapenemase target were used. Results: Each one of the two carbapenemase gene tested presented a different melting curve after PCR amplification. The melting temperature (Tm) analysis of the amplicons identified was as follows: blaIMP type (Tm 82.18°C), blaNDM-1 (Tm 78.8°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. Conclusions: The new assay was able to detect the presence of two different carbapenemase gene type by real-time PCR.

Keywords: resistance, b-lactamases, E. coli, real-time PCR

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Authors: Meriche Hacene

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Introduction: Celiac disease (CD) is a chronic immune-mediated enteropathy triggered by gluten found in wheat or oats or rye. Celiac disease is associated with the HLA-DQ2 and HLA-DQ8 susceptibility alleles. This association with the HLA DQ2/DQ8 molecules confirmed the responsibility of genetic factors that intervene in the triggering of the autoimmune process of this condition. Objective: To evaluate the results of HLA DQ2 and HLA DQ8 typing of 40 patients suspected of having CD by PCR-SSP (Polymerase Chain Reaction Sequence Specific Primers). Material and method : 40 patients suspected of celiac disease with IgA transglutaminase serology (-) and duodenal biopsy (+). HLADR/DQ PCR-SSP (fluogen-innotrain) typing was carried out. Results : The average age of adults was 40 years, children: 4 years, the sex ratio was 1M/3F. In our patients the HLA DQ2 allele is found with a frequency of 75%, the DQ8 with a frequency of 25%, 17.5% were HLA-DQ2 homozygous and 15% were HLADQ2/HLADQ8. In our series, HLADQ2, DQ8 are found in almost all patients with a frequency of 95%. 30% of patients in our study had associated positivity of HLA-DRB3, DRB4 or DRB5 alleles. Conclusion : A high prevalence of positivity of HLADQ2 alleles at the expense of HLA DQ8 was found, which is consistent with literature data. These molecules constitute an additional marker for screening and diagnosis of CD.

Keywords: HLA typing, coeliac disease, HLA DQ 2, HLA DQ8

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Authors: Zishan Ahmad, Anwar Shahzad

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The present studies describe a protocol for high frequency in vitro propagation through nodal segments and shoot tips in D. arayalpathra, a critically endangered medicinal liana of the Western Ghats, India. Nodal segments were more responsive than shoot tips in terms of shoot multiplication. Murashige and Skoog’s (MS) basal medium supplemented with 2.5 µM 6-benzyladenine (BA) was optimum for shoot induction through both the explants. Among different combinations of plant growth regulator (PGRs) and growth additive screened, MS medium supplemented with BA (2.5 µM) + indole-3-acetic acid (IAA) (0.25 µM) + adenine sulphate (ADS) (10.0 µM) induced a maximum of 9.0 shoots per nodal segment and 3.9 shoots per shoot tip with mean shoot length of 8.5 and 3.9 cm respectively. Half-strength MS medium supplemented with Naphthaleneacetic acid (NAA) (2.5 µM) was the best for in vitro root induction. After successful acclimatization in SoilriteTM, 92 % plantlets were survived in field conditions. Acclimatized plantlets were studied for chlorophyll and carotenoid content, net photosynthetic rate (PN) and related attributes such as stomatal conductance (Gs) and transpiration rate during subsequent days of acclimatization. The rise and fall of different biochemical enzymes (SOD, CAT, APX and GR) were also studies during successful days of acclimatization. Moreover, the effect of acclimatization on the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2H4MB) was also studied in relation to the biomass production. Maximum fresh weight (2.8 gm/plant), dry weight (0.35 gm/plant) of roots and 2H4MB content (8.5 µg/ ml of root extract) were recorded after 8 weeks of acclimatization. The screening of in vitro raised plantlet root was also carried out by using GC-MS analysis which witnessed more than 25 compounds. The regenerated plantlets were also screened for homogeneity by using RAPD and ISSR. The proposed protocol surely can be used for the conservation and commercial production of the plant.

Keywords: 6-benzyladenine, PGRs, RAPD, 2H4MB

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Authors: Abeer M. Algeblawi

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Fifteen isolates of Agrobacterium tumefaciens were obtained from crown gall samples collected from six locations (Tripoli, Alzahra, Ain-Zara, Alzawia, Alazezia in Libya) from Grape (Vitis vinifera L.), Pear (Pyrus communis L.), Peach (Prunus persica L.) and Alexandria in Egypt from Guava (Psidium guajava L.) trees, Artichoke (Cynara cardunculus L.) and Sugar beet (Beta vulgaris L.). Total DNA was extracted from the eight isolates as well as the identification of six isolates used into Polymerase Chain Reaction (PCR) analysis and Random Amplified Polymorphic DNA (RAPD) technique were used. High similarity (55.5%) was observed among the eight A. tumefaciens isolates (Agro1, Agro2, Agro3, Agro4, Agro5, Agro6, Agro7, and Agro8). The PCR amplification products were resulting from the use of two specific primers (virD2A-virD2C). Analysis induction six isolates of A. tumefaciens obtained from different hosts. A visible band was specific to A. tumefaciens of (220 bp, 224 bp) and 338 bp produced with total DNA extracted from bacterial cells.

Keywords: Agrobacterium tumefaciens, crown gall, identification, molecular characterization, PCR, RAPD

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Authors: Borys Stegniy, Anton Gerilovych, Oleksii Solodiankin, Vitaliy Bolotin, Anton Stegniy, Denys Muzyka, Claudio Afonso

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New APMV was isolated from white fronted goose in Ukraine. This isolate was tested serologically using monoclonal antibodies in haemagglutination-inhibition tests against APMV1-9. As the results obtained isolate showed cross reactions with APMV7. Following investigations were provided for the full genome sequencing using random primers and cloning into pCRII-TOPO. Analysis of 100 transformed colonies of E.coli using traditional sequencing gave us possibilities to find only 3 regions, which could identify by BLAST. The first region with the length of 367 bp had 70 % nucleotide sequence identity to the APMV 12 isolate Wigeon/Italy/3920_1/2005 at genome position 2419-2784. Next region (344 bp) had 66 % identity to the same APMV 12 isolate at position 4760-5103. The last region (365 bp) showed 71 % identity to Newcastle disease virus strain M4 at position 12569-12928.

Keywords: APMV, Newcastle disease virus, Ukraine, full genome sequencing

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Authors: Prachi Sharma, Mukesh Kumar Rana

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In the present study, the simple sequence repeat(SSR) markers have been used in analysis of genetic diversity of 37 genotypes of Triticum aestivum. The DNA was extracted using cTAB method. The DNA was quantified using the fluorimeter. The annealing temperatures for 27 primer pairs were standardized using gradient PCR, out of which 16 primers gave satisfactory amplification at temperature ranging from 50-62⁰ C. Out of 16 polymorphic SSR markers only 10 SSR primer pairs were used in the study generating 34 reproducible amplicons among 37 genotypes out of which 30 were polymorphic. Primer pairs Xgwm533, Xgwm 160, Xgwm 408, Xgwm 120, Xgwm 186, Xgwm 261 produced maximum percent of polymorphic bands (100%). The bands ranged on an average of 3.4 bands per primer. The genetic relationship was determined using Jaccard pair wise similarity co-efficient and UPGMA cluster analysis with NTSYS Pc.2 software. The values of similarity index range from 0-1. The similarity coefficient ranged from 0.13 to 0.97. A minimum genetic similarity (0.13) was observed between VL 804 and HPW 288, meaning they are only 13% similar. More number of available SSR markers can be useful for supporting the genetic diversity analysis in the above wheat genotypes.

Keywords: wheat, genetic diversity, microsatellite, polymorphism

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Authors: Ilian Badjakov, Ivayla Dincheva, Violeta Kondakova, Rossitza Batchvarova

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In this study, we present our initial results on the assessment of genetic diversity among wild Bulgarian berry accessions (Rubus idaeus L. Fragaria Vesca L., Vaccinium vitis-idaea L., Vaccinium myrtillus L.) using Random Amplified Polymorphic DNA (RAPDs) markers. Leaves and fruits were collected from two natural habitats - the Balkan Mountain and the Mountain of Orpheus - Rhodope Mountain. All accessions were screened for their polymorphism using five RAPD primers. The phylogenetic distances calculated from RAPD data ranged from 0.29 to 0.82 thus indicating that a high level of gene diversity is present in the selected genotypes. In order to characterize further the structure and grouping of berry accessions, a dendrogram deriving from UPGMA cluster analysis based on the genetic similarity (GS) coefficient matrix was designed. RAPD analysis provided to be efficient for discrimination of accessions within the same species with similar morphological characters

Keywords: Bulgarian wild berries, genetic diversity, RAPD, UPGMA

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Authors: Faheema Khan

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To investigate the ability of sensitive and tolerant genotype of Glycine max to adapt to a saline environment in a field, we examined the growth performance, water relation and activities of antioxidant enzymes in relation to photosynthetic rate, chlorophyll a fluorescence, photosynthetic pigment concentration, protein and proline in plants exposed to salt stress. Ten soybean genotypes (Pusa-20, Pusa-40, Pusa-37, Pusa-16, Pusa-24, Pusa-22, BRAGG, PK-416, PK-1042, and DS-9712) were selected and grown hydroponically. After 3 days of proper germination, the seedlings were transferred to Hoagland’s solution (Hoagland and Arnon 1950). The growth chamber was maintained at a photosynthetic photon flux density of 430 μmol m−2 s−1, 14 h of light, 10 h of dark and a relative humidity of 60%. The nutrient solution was bubbled with sterile air and changed on alternate days. Ten-day-old seedlings were given seven levels of salt in the form of NaCl viz., T1 = 0 mM NaCl, T2=25 mM NaCl, T3=50 mM NaCl, T4=75 mM NaCl, T5=100 mM NaCl, T6=125 mM NaCl, T7=150 mM NaCl. The investigation showed that genotype Pusa-24, PK-416 and Pusa-20 appeared to be the most salt-sensitive. genotypes as inferred from their significantly reduced length, fresh weight and dry weight in response to the NaCl exposure. Pusa-37 appeared to be the most tolerant genotype since no significant effect of NaCl treatment on growth was found. We observed a greater decline in the photosynthetic variables like photosynthetic rate, chlorophyll fluorescence and chlorophyll content, in salt-sensitive (Pusa-24) genotype than in salt-tolerant Pusa-37 under high salinity. Numerous primers were verified on ten soybean genotypes obtained from Operon technologies among which 30 RAPD primers shown high polymorphism and genetic variation. The Jaccard’s similarity coefficient values for each pairwise comparison between cultivars were calculated and similarity coefficient matrix was constructed. The closer varieties in the cluster behaved similar in their response to salinity tolerance. Intra-clustering within the two clusters precisely grouped the 10 genotypes in sub-cluster as expected from their physiological findings.Salt tolerant genotype Pusa-37, was further analysed by 2-Dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the Present study, 173 protein spots were identified. Of these, 40 proteins responsive to salinity were either up- or down-regulated in Pusa-37. Proteomic analysis in salt-tolerant genotype (Pusa-37) led to the detection of proteins involved in a variety of biological processes, such as protein synthesis (12 %), redox regulation (19 %), primary and secondary metabolism (25 %), or disease- and defence-related processes (32 %). In conclusion, the soybean plants in our study responded to salt stress by changing their protein expression pattern. The photosynthetic, biochemical and molecular study showed that there is variability in salt tolerance behaviour in soybean genotypes. Pusa-24 is the salt-sensitive and Pusa-37 is the salt-tolerant genotype. Moreover this study gives new insights into the salt-stress response in soybean and demonstrates the power of genomic and proteomic approach in plant biology studies which finally could help us in identifying the possible regulatory switches (gene/s) controlling the salt tolerant genotype of the crop plants and their possible role in defence mechanism.

Keywords: glycine max, salt stress, RAPD, genomic and proteomic variability

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Authors: T. C. C. Soo, S. Bhassu

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Unlike the common presence of both innate and adaptive immunity in vertebrates, crustaceans, in particular, shrimps, have been discovered to possess only innate immunity. This further emphasizes the importance of innate immunity within shrimps in pathogenic resistance. Under the study of pathogenic immune challenge, different shrimp species actually exhibit varying degrees of immune resistance towards the same pathogen. Furthermore, even within the same shrimp species, different batches of challenged shrimps can have different strengths of immune defence. Several important pathways are activated within shrimps during pathogenic infection. One of them is JAK-STAT pathway that is activated during bacterial, viral and fungal infections by which STAT(Signal Transducer and Activator of Transcription) gene is the core element of the pathway. Based on theory of Central Dogma, the genomic information is transmitted in the order of DNA, RNA and protein. This study is focused in uncovering the important evolutionary patterns present within the DNA (non-coding region) and RNA (coding region). The three shrimp species involved are Macrobrachium rosenbergii, Penaeus monodon and Litopenaeus vannamei which all possess commercial significance. The shrimp species were challenged with a famous penaeid shrimp virus called white spot syndrome virus (WSSV) which can cause serious lethality. Tissue samples were collected during time intervals of 0h, 3h, 6h, 12h, 24h, 36h and 48h. The DNA and RNA samples were then extracted using conventional kits from the hepatopancreas tissue samples. PCR technique together with designed STAT gene conserved primers were utilized for identification of the STAT coding sequences using RNA-converted cDNA samples and subsequent characterization using various bioinformatics approaches including Ramachandran plot, ProtParam and SWISS-MODEL. The varying levels of immune STAT gene activation for the three shrimp species during WSSV infection were confirmed using qRT-PCR technique. For one sample, three biological replicates with three technical replicates each were used for qRT-PCR. On the other hand, DNA samples were important for uncovering the structural variations within the genomic region of STAT gene which would greatly assist in understanding the STAT protein functional variations. The partially-overlapping primers technique was used for the genomic region sequencing. The evolutionary inferences and event predictions were then conducted through the Bayesian Inference method using all the acquired coding and non-coding sequences. This was supplemented by the construction of conventional phylogenetic trees using Maximum likelihood method. The results showed that adaptive evolution caused STAT gene sequence mutations between different shrimp species which led to evolutionary divergence event. Subsequently, the divergent sites were correlated to the differing expressions of STAT gene. Ultimately, this study assists in knowing the shrimp species innate immune variability and selection of disease resistant shrimps for breeding purpose. The deeper understanding of STAT gene evolution from the perspective of both purifying and adaptive approaches not only can provide better immunological insight among shrimp species, but also can be used as a good reference for immunological studies in humans or other model organisms.

Keywords: gene evolution, JAK-STAT pathway, immunology, STAT gene

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Authors: Abdelsabour G. A. Khaled, Ahmed W. A. Abdalla And Sabry Y. M. Mahmoud

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Banana plants showing typical mosaic and yellow stripes on leaves as symptoms were collected from Assiut Governorate in Egypt. The causal agent was identified as Cucumber mosaic virus (CMV) on the basis of symptoms, transmission, serology, transmission electron microscopy and reverse transcription polymerase chain reaction (RT-PCR). Coat protein (CP) gene was amplified using gene specific primers for coat protein (CP), followed by cloning into desired cloning vector for sequencing. In this study the CMV was transmitted into propagation host either by aphid or mechanically. The transmission was confirmed through Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA). Analysis of the 120 deduced amino acid sequence of the coat protein gene revealed that the EG-A strain of CMV shared from 97.50 to 98.33% with those strains belonging to subgroup IA. The cluster analysis grouped the Egyptian isolate with strains Fny and Ri8 belonging sub-group IA. It appears that there occurs a high incidence of CMV infecting banana belonging to IA subgroup in most parts of Egypt.

Keywords: banana, CMV, transmission, CP gene, RT-PCR

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Authors: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

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Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

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Authors: Nassibeh Hosseini-Vasoukolaei, Amir Ahmad Akhavan, Mahmood Jeddi-Tehrani, Ali Khamesipour, Mohammad Reza Yaghoobi Ershadi, Kamhawi Shaden, Valenzuela Jesus, Hossein Mirhendi, Mohammad Hossein Arandian

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Zoonotic cutaneous leishmaniasis (ZCL) is an endemic disease in many rural areas of Iran. Sand flies were collected from rural areas of Esfahan province and were identified using valid identification keys. DNA was extracted from sand flies and Nested PCRs were done using specific primers. In this study, 44 out of 152 (28.9 %) sand flies were infected with L. majoralone. Eight sand flies showed mixed infection: four sand flies (2.6 %) were infected with L. major, L. turanicaand L. gerbili, one sand fly (0.7 %) was infected with L. major and L. turanica and three sand flies (2 %) were infected with L. turanicaand L. gerbili. Our results demonstrate the natural infection of P. papatasi sand fly with three species of L. major, L. turanica and L. gerbili which are circulating among R. opimusreservoir host and P. papatasi sand fly vector in central Iran.

Keywords: Phlebotomus papatasi, Leishmania major, Leishmania turanica, Leishmania gerbili, mixed infection, Iran

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Authors: Aptin Rahnavard, Ghavamaldin Asadian, Khalil Pourshamsian, Mariamalsadat Taghavi

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Dog rose is one of the important rose species in Iran that the distant past had been considered due to nutritional value and medicinal. Despite its long history of use, due to poor information on the genetic modification of plants has been done resources inheritance. In this study was to assess the genetic diversity. Total of 30 genotypes Dog rose from areas of northern Iran in the Caspian region (provinces of Guilan and Mazandaran) were evaluated using 25 RAPD primers. The number of bands produced total of 202 and for each primer were measured in a bands with an average 8-band .The number of polymorphic bands per primer ranged from 1 to 13 and the bands were in the range of 300 to 3000 bp. Based on the results OPA-04 primer with 13 bands and PRA-1, E-09 and A-04 with 5-band were created maximum and minimum number of amplified fragments. Molecular marker genotypes showed a high degree of polymorphism. Studied genotypes based on RAPD results were divided into 2 groups and 2 subgroups. Most similar in subgroups A2 and B group was the lowest.

Keywords: rosa canina spp., RAPD marker, genetic variation, caspian climate

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Authors: Masoud Alipanah, Sekineh Akbari, Gholamreza Dashab

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Myostatin genes or factor 8 affecting on growth and making differentiation works (GDF8) as a moderator in the development of skeletal muscle inhibitor. If mutations occurs in the coding region of myostatin, alter its inhibitory role and the muscle growth is increased. In this study, blood samples were collected randomly from 60 Kurdish sheep in northern Khorasan and DNA extraction was performed using a modified salt. A fragment 337 bp from exon 3 myostatin gene and-specific primers by using a polymerase chain reaction (PCR) were amplified. In order to detect different forms of an allele at this locus HaeΙΙΙ restriction enzymes and PCR-RFLP analysis were used. Band patterns clarification was performed using agarose gel electrophoresis. The frequency of genotypes mm, Mm, and MM, were respectively detected, 0, 0.15 and 0.85. The allele frequency for alleles m and M, were respectively, 0.07 and 0.93. The statistical analyses indicated that m allele was significantly associated with body weight. The results of this study suggest that the Myostatin gene possibly is a candidate gene that affects growth traits in Kurdish sheep.

Keywords: GDF8 gene, Kurdi Sheep of Northern Khorasan, polymorphism, weight traits

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Authors: Ali Seirafi

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The purpose of this study was to investigate the effect and application of starch nanocrystals on physicochemical and microbial properties in the industrial production of probiotic yogurt. In this study, probiotic yoghurt was manufactured by industrial method with the optimization and control of the technological factors affecting the probabilistic biomass, using probiotic bacteria Lactobacillus acidophilus and Bifidobacterium bifidum with commonly used yogurt primers. Afterwards, the effects of different levels of fat (1.3%, 2.5 and 4%), as well as the effects of various perbiotic compounds include starch nanocrystals (0.5%, 1 and 1.5%), galactolegalosaccharide (0.5% 1 and 1.5%) and fructooligosaccharide (0.5%, 1 and 1.5%) were evaluated. In addition, the effect of packaging (polyethylene and glass) was studied, while the effect of pH changes and final acidity were studied at each stage. In this research, all experiments were performed in 3 replications and the results were analyzed in a completely randomized design with SAS version 9.1 software. The results of this study showed that the addition of starch nanocrystal compounds as well as the use of glass packaging had the most positive effects on the survival of Lactobacillus acidophilus bacteria and the addition of nano-crystals and the increase in the cooling rate of the product, had the most positive effects on the survival of bacteria Bifidobacterium bifidum.

Keywords: Bifidobacterium bifidum, Lactobacillus acidophilus, prebiotics, probiotic yogurt

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Authors: Ahdieh Alijani, Mina Zarrabi, Abdollah Jamshidi, Jamshid Razmyar

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Avian intestinal spirochetosis (AIS) is caused by spiral-shaped Gram-negative Brachyspira spp. in poultry and is known as a cause of diarrhea, low egg production and increased the occurrence of dirty eggs in layer hens. In this study the presence of some Brachyspira spp. was investigated in laying hens. A total of 100 cloacal swab samples were individually collected from 20 laying hen flocks showing fecal egg staining in northeastern Iran. By culture and morphologic examination, 41 samples (41%) from 20 flocks were positive but by using genus–specific PCR only 37 (37%) samples were confirmed as Brachyspira spp. Using species-specific primers, single colonization was identified in 18 samples associated with B. pilosicoli (48.6%) while single colonization with B. intermedia was found in only two samples (5.4%). Simultaneous colonization by B. intermedia and B. murdochii was detected in 3 samples (8.1%). B. pilosicoli was the most prevalent species in concurrent colonization in 11 cases (29.7%). Finally, co-colonization by B. intermedia and B. innocens was identified in 3 samples (8.1%). The results of this study show the colonization of different species of Brachyspira with the dominance of B. pilosicoli in layer hens. In simultaneous colonization with pathogenic and non-pathogenic species the symptoms of intestinal spirochetosis were reduced, suggesting a competitive role in preventing and reducing the colonization of pathogenic species.

Keywords: intestinal spirochetosis, Brachyspira, laying hen, PCR

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Authors: Ahreum Choi, Otgontuya Tsogbadrakh, Kwang-Hwan Jung

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Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied. These retinal-binding proteins have divided into two types. The type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. For type I rhodopsin, there are two main functions that are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is one of the microbial rhodopsin with main function as photo-sensory transduction. Although ASR is expressed fairly well in Escherichia coli, the expression level is relatively less compare to Proteorhodopsin. In this study, full length of ASR was used to test for the expression influence by codon usage in E. coli. Eight amino acids of codon at N-terminal part of ASR were changed randomly with designed primers, which allow 8,192 nucleotide different cases. The codon changes were screened for the preferable codons of each residue, which have given higher expression yield. Among those 57 selected mutations, there are 24 color-enhanced E. coli colonies that contain ASR proteins, and it showed better expression level than the wild type ASR codon usage. This strongly suggests that high codon usage of only partial N-terminal of protein can increase the expression level of whole protein.

Keywords: 7-transmembrane, all-trans retinal, rhodopsin, codon-usage, protein expression

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Authors: Md. Baki Billah, Suraiya Parveen, Shuvra Kanti Dey

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Bangladesh is earning a lot of foreign currency by exporting shrimp, but this industry is facing a tremendous problem due to the infection of white spot syndrome virus (WSSV). This study was undermined to develop rapid detection method of WSSV. A total of shrimp samples 240 collected from the 12 shrimp farms of different coastal regions (Satkhira, Khulna, and Bagerhat) were analyzed by conventional PCR using VP28 and VP664 gene-specific primers. In satkhira, Bagerhat and Khulna 39, 41 and 29 samples were found WSSV positive respectively. Real-time PCR using 71-bp amplicon for VP664 gene correlated well with conventional PCR data. The prevalence rates of WSSV among the collected 240 samples were Satkhira 38%, Khulna 47% and Bagerhat 50%. Molecular analysis of the VP28 gene sequences of WSSV revealed that Bangladeshi strains phylogenetically affiliated to the strains belong to India. This work concluded that WSSV infections are widely distributed in the coastal regions cultured shrimp in Bangladesh. Physico-chemical parameters were within the range of fish culture.

Keywords: coastal regions of Bangladesh, PCR, shrimp, white spot syndrome virus

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Authors: J. Bernasovská, R. Lohajová Behulová, E. Petrejčiková, I. Boroňová, I. Bernasovský

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Y chromosome microdeletions are the most common genetic cause of male infertility and screening for these microdeletions in azoospermic or severely oligospermic men is now standard practice. Analysis of the Y chromosome in men with azoospermia or severe oligozoospermia has resulted in the identification of three regions in the euchromatic part of the long arm of the human Y chromosome (Yq11) that are frequently deleted in men with otherwise unexplained spermatogenic failure. PCR analysis of microdeletions in the AZFa, AZFb and AZFc regions of the human Y chromosome is an important screening tool. The aim of this study was to analyse the type of microdeletions in men with fertility disorders in Slovakia. We evaluated 227 patients with azoospermia and with normal karyotype. All patient samples were analyzed cytogenetically. For PCR amplification of sequence-tagged sites (STS) of the AZFa, AZFb and AZFc regions of the Y chromosome was used Devyser AZF set. Fluorescently labeled primers for all markers in one multiplex PCR reaction were used and for automated visualization and identification of the STS markers we used genetic analyzer ABi 3500xl (Life Technologies). We reported 13 cases of deletions in the AZF region 5,73%. Particular types of deletions were recorded in each region AZFa,b,c .The presence of microdeletions in the AZFc region was the most frequent. The study confirmed that percentage of microdeletions in the AZF region is low in Slovak azoospermic patients, but important from a prognostic view.

Keywords: AZF, male infertility, microdeletions, Y chromosome

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Authors: Fayyaz Rasool, Shakeela Parveen

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The studies on genetic diversity of Labeo rohita by using molecular markers were carried out to investigate the genetic structure by RAPAD marker and the levels of polymorphism and similarity amongst the different groups of five populations of wild and farmed types. The samples were collected from different five locations as representatives of wild and hatchery raised populations. RAPAD data for Jaccard’s coefficient by following the un-weighted Pair Group Method with Arithmetic Mean (UPGMA) for Hierarchical Clustering of the similar groups on the basis of similarity amongst the genotypes and the dendrogram generated divided the randomly selected individuals of the five populations into three classes/clusters. The variance decomposition for the optimal classification values remained as 52.11% for within class variation, while 47.89% for the between class differences. The Principal Component Analysis (PCA) for grouping of the different genotypes from the different environmental conditions was done by Spearman Varimax rotation method for bi-plot generation of the co-occurrence of the same genotypes with similar genetic properties and specificity of different primers indicated clearly that the increase in the number of factors or components was correlated with the decrease in eigenvalues. The Kaiser Criterion based upon the eigenvalues greater than one, first two main factors accounted for 58.177% of cumulative variability.

Keywords: variation, clustering, PCA, wild, hatchery, RAPAD, Labeo rohita

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Authors: Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman, Mohd Sukri Hassan

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Real-time PCR is a molecular biology technique that is currently being widely used for halal services to differentiating between porcine and bovine DNA. The useful of technique become very important for student or workers (who works in the laboratory) to learn how the technique could be run smoothly without fail. Same concept with conventional PCR, real-time PCR also needed DNA template, primer, enzyme polymerase, dNTP, and buffer. The difference is in real-time PCR, have additional component namely fluorescent dye. The most common use of fluorescent dye in real-time PCR is SYBR green. The purpose of this study was to find out how sensitive, specific and efficient real-time PCR technique was combined with SYBR green method and specific primers of CYT b. The results showed that real-time PCR technique using SYBR Green, capable of detecting porcine and bovine DNA concentrations up to 0.0001 µl/ng. The level of efficiency for both types of DNA was 91% (90-110). Not only that in specific primer CYT b bovine primer could detect only bovine DNA, and porcine primer could detect only porcine primer. So, from the study could be concluded that real-time PCR technique that was combined with specific primer CYT b and SYBR green method, was sensitive, specific and efficient to detect porcine and bovine DNA.

Keywords: sensitivity, specificity, efficiency, real-time PCR, SYBR green, Cytochrome b, porcine DNA, bovine DNA

Procedia PDF Downloads 288

Authors: Hiba Dakroub, Danilo Russo, Luca Cistrone, Francesco Serra, Giovanna Fusco, Esterina De Carlo, Maria Grazia Amoroso

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The question of the origin of SARS-CoV-2 and the cycle of transmission between humans and animals is still unanswered. One serious concern associated with the SARS-CoV-2 pandemic is that the virus might spill back from humans to wildlife, which would render some animal species reservoirs of the human virus. The aim of the present study is to monitor the potential risk of SARS-CoV-2 reverse infection from humans to bats, by performing bat surveillance from different sites in Central-Southern Italy. We collected 240 droppings or saliva from 129 bats and tested them using specific and general primers of SARS-COV-2 and coronaviruses respectively. All samples, including 127 nasal swabs and 113 fecal droppings resulted negative for SARS-COV-2, and these results were confirmed by testing the samples with the Droplet Digital PCR. Also, an end-point RT-PCR was performed and no sample showed specific bands. The absence of SARS-CoV-2 in the bats we surveyed is a first step towards a better understanding of reverse transmission to bats of this virus. We hope our first contribution will encourage the establishment of systematic surveillance of wildlife, and specifically bats, to help prevent reverse zoonotic episodes that would jeopardize human health as well as biodiversity conservation and management.

Keywords: coronaviruses, bats, zoonotic viruses, spillback, SARS-CoV-2

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Authors: Shokoufeh Roudashti, Shahin Bahari, Fakhri Haghi, Habib Zeighami, Ghazal Naderi, Paniz Shirmast

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Background: Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB) in humans and animals. Mycobacterium MTBC is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The disease can transmit to human by direct contact with the infected animals, drinking unpasteurized milk and consumption of uncooked meat. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk. Tuberculosis MTBC is the predominant infectious cause of morbidity and morality worldwide, It is estimated that one third of the world population (approx. 1.8 billion persons) is infected with M. tuberculosis and each year there are 8 million new cases worldwide. The aim of this study, to detect Mycobacterium MTBC in raw milk samples using polymerase chain reaction (PCR). Materials and Methods: In the present study, 60 raw milk samples were collected from rural areas in Zanjan, Iran. After extraction of DNAs and using special primers for Is6110 gene as a marker, PCR was applied to detect the presence or non-presence of the related gene. Results: According to the findings of this study, 8 (13.5 %) out of 60 milk samples were positive for Mycobacterium spp (P < 0.1). Conclusions: The Outbreak of genus Mycobacteria spp in milk samples were determined to be relatively high in Zanjan, Iran.

Keywords: Mycobacteria spp, raw milk, PCR, Zanjan

Procedia PDF Downloads 265

Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

Procedia PDF Downloads 309