Search results for: primer sequences

Authors: Thu Thuy Pham, Thi Chau Loan Tran, Van Duy Nguyen

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Marine fungi play an important role in the marine ecosystems. Marine fungi also supply biomass and metabolic products of industrial value. Currently, the biodiversity of marine fungi along the coastal areas of Vietnam has not yet been studied fully. The objective of this study is to assess the spatial and temporal diversity of planktonic fungi from the coastal waters of Nha Trang Bay and Van Phong Bay in Central Vietnam using culture-dependent and independent approach. Using culture-dependent approach, filamentous fungi and yeasts were isolated on selective media and then classified by phenotype and genotype based on the sequencing of ITS (internal transcribed spacers) regions of rDNA with two primer pairs (ITS1F_KYO2 and ITS4; NS1 and NS8). Using culture-independent approach, environmental DNA samples were isolated and amplified using fungal-specific ITS primer pairs. A total of over 160 strains were isolated from 10 seawater sampling stations at 50 cm depth. They were classified into diverse genera and species of both yeast and mold. At least 5 strains could be potentially novel species. Our results also revealed that planktonic fungi were molecularly diverse with hundreds of phylotypes recovered across these two bays. The results of the study provide data about the distribution and taxonomy of mycoplankton in this area, thereby allowing assessment of their positive role in the biogeochemical cycle of coastal ecosystems and the development of new bioactive compounds for industrial applications.

Keywords: biodiversity, ITS, marine fungi, Nha Trang Bay, Van Phong Bay

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Authors: Jung Ho Yang, Yong Soo Kim

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Electrical units are operated by electrical and electronic components. An environmental test sequence is useful for testing electrical units to reduce reliability issues. This study introduces test sequence guidelines based on relevant principles and considerations for electronic testing according to international standard IEC-60068-1 and the United States military standard MIL-STD-810G. Then, test sequences were proposed based on the descriptions for each test. Finally, General Motors (GM) specification GMW3172 was interpreted and compared to IEC-60068-1 and MIL-STD-810G.

Keywords: reliability, environmental test sequence, electrical units, IEC 60068-1, MIL-STD-810G

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Authors: Kalyan S. Ghosh, B. L. Dhananjaya

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Snake bite causes fatal injuries in multi-organs and even many deaths due to several adverse physiological effects of various phospholipases (PLA2s) present in snake venom. Though these PLA2s bear highly homologues sequences and also structure but exhibit a different extent of those pharmacological effects. In this study, we have explored the difference in the neurotoxicity of two PLA2 namely PLA2-V, PLA2-VIIIa present in the venom from Vipera russellii. Bioinformatics studies on sequences of these two proteins along with detailed structural comparison enable us to explore the differences unambiguously. The identification of the residues involved in neurotoxicity will further lead towards proper designing of inhibitors against such killing effects of the venom.

Keywords: electrostatic potential, homology modeling, hydrophobicity, neurotoxicity, Phospholipase A2

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Authors: Muhammad Fiaz Qamar, Ahmad Faraz, Muhammad Arfan Zaman, Kazim Ali, Waleed Akram

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Babesiosis is reflected as the most important disease of cattle that are transmitted by arthropods. In Pakistan, its prevalence is up to 29% in the cattle and buffalo population in different regions. Cattle show a long lasting and durable immunity by giving an infection of B.bovis, B. bigemina, or Babesiadivergens. this is used in cattle to immunize them in a few countries as anti-babesiosis vaccine. Development of frozen vaccine allows for complete testing after production of each batch, However, once thawed, its reduced its shelf life, frozen vaccines are more difficult to transport as well as expensive to produce as compared to chilled vaccine. The contamination of blood derived vaccine has the potential risk that makes pre-production and post-production quality control necessary. For the trail master seed production of whole blood frozen bivalent Babesia(Babesiabovis and Babesiabigemina), 100 blood samples of Babesial positive suspected cattle was taken and processed for separation microscopic detection and rectification by PCR. Vaccine passages were done to reduce the parasitaemiasis in live calves. After 8 passages, parasitemia of Babesia reduced from 80% to 15%. Infected donor calf’s blood was taken by jugular cannulation by using preservative free lithium heparin as an anticoagulant (5 International Units IU heparin/ml blood). In lab, parasite containing blood was mixed in equal volumes with 3 M glycerol in PBS supplemented with 5 mM glucose (final concentration of glycerol 1.5 M) at 37°C. The mixture was then equilibrized at 37°C for 30 minutes and were dispensed in required containers (e.g., 5 ml cryovials).

Keywords: distribution, babesia, primer sequences, PCV

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Authors: Shahrzad Mohammadi, Ghasem Boshrouei Sharq

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All seismic design codes are based on the determination of the design earthquake without taking into account the effects of aftershocks in the design practice. In regions with a high level of seismicity, the occurrence of several aftershocks of various magnitudes and different time lags is very likely. This research aims to estimate the collapse capacity of a 10-story steel bundled tube moment frame subjected to as-recorded seismic sequences. The studied structure is designed according to the seismic regulations of the fourth revision of the Iranian code of practice for the seismic-resistant design of buildings (Code No.2800). A series of incremental dynamic analyses (IDA) is performed up to the collapse level of the intact structure. Then, in order to demonstrate the effects of aftershock events on the collapse vulnerability of the building, aftershock IDA analyzes are carried out. To gain deeper insight, collapse fragility curves are developed and compared for both series. Also, a study on the influence of various ground motion characteristics on collapse capacity is carried out. The results highlight the importance of considering the decisive effects of aftershocks in seismic codes due to their contribution to the occurrence of collapse.

Keywords: IDA, aftershock, bundled tube frame, fragility assessment, GM characteristics, as-recorded seismic sequences

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Authors: Kyung Jin Kim, Jiwon Kwak, Jae-Hoon Lee, Soo Suk Lee

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We describe an innovative method for highly specific detection of miRNAs using a specially modified method of poly(A) adaptor RT-qPCR. We use uniquely designed specific extension sequence, which plays important role in providing an opportunity to affect high specificity of miRNA detection. This method involves two steps of reactions as like previously reported and which are poly(A) tailing and reverse-transcription followed by real-time PCR. Firstly, miRNAs are extended by a poly(A) tailing reaction and then converted into cDNA. Here, we remarkably reduced the reaction time by the application of short length of poly(T) adaptor. Next, cDNA is hybridized to the 3’-end of a specific extension sequence which contains miRNA sequence and results in producing a novel PCR template. Thereafter, the SYBR Green-based RT-qPCR progresses with a universal poly(T) adaptor forward primer and a universal reverse primer. The target miRNA, miR-106b in human brain total RNA, could be detected quantitatively in the range of seven orders of magnitude, which demonstrate that the assay displays a dynamic range of at least 7 logs. In addition, the better specificity of this novel extension-based assay against well known poly(A) tailing method for miRNA detection was confirmed by melt curve analysis of real-time PCR product, clear gel electrophoresis and sequence chromatogram images of amplified DNAs.

Keywords: microRNA(miRNA), specific extension sequence, RT-qPCR, poly(A) tailing assay, reverse transcription

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Authors: Balaji Ganesh Rajagopal, Subramanian Appavu alias Balamurugan, Ayyalraj Midhun Kumar, Krishnan Nallaperumal

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Intelligent Transportation System is an Emerging area to solve multiple transportation problems. Several forms of inputs are needed in order to solve ITS problems. Advanced Traveler Information System (ATIS) is a core and important ITS area of this modern era. This involves travel time forecasting, efficient road map analysis and cost based path selection, Detection of the vehicle in the dynamic conditions and Traffic congestion state forecasting. This Article designs and provides an algorithm for traffic data generation which can be used for the above said ATIS application. By inputting the real world traffic situation in the form of video sequences, the algorithm determines the Traffic density in terms of congestion, number of vehicles in a given path which can be fed for various ATIS applications. The Algorithm deduces the key frame from the video sequences and follows the Blob detection, Identification and Tracking using connected components algorithm to determine the correlation between the vehicles moving in the real road scene.

Keywords: traffic transportation, traffic density estimation, blob identification and tracking, relative velocity of vehicles, correlation between vehicles

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Authors: Yijun Lai, Saber Khederzadeh, Lingshaung Han

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Species in Thunnus are organized due to the similarity between them. The closeness between T. maccoyii, T. thynnus, T. Tonggol, T. atlanticus, T. albacares, T. obsesus, T. alalunga, and T. orientails are in different degrees. However, the genetic pattern of differentiation has not been presented based on individuals yet, to the author’s best knowledge. Hence, we aimed to analyze the difference in individuals level of tuna species to identify the factors that contribute to the maternal lineage variety using Cytochrome c oxidase subunit I (COXI) gene sequences. Our analyses provided evidence of sharing lineages in the Thunnus. A phylogenetic analysis revealed that these lineages are basal to the other sequences. We also showed a close connection between the T. tonggol, T. thynnus, and T. albacares populations. Also, the majority of the T. orientalis samples were clustered with the T. alalunga and, then, T. atlanticus populations. Phylogenetic trees and migration modeling revealed high proximity of T. thynnus sequences to a few T. orientalis and suggested possible gene flow with T. tonggol and T. albacares lineages, while all T. obsesus samples indicated unique clustering with each other. Our results support the presence of old maternal lineages in Thunnus, as a legacy of an ancient wave of colonization or migration.

Keywords: Thunnus Tuna, phylogeny, maternal lineage, COXI gene

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Authors: Nkwanyang L. Takem, Christopher M. Agyingi

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Background and aim: An integrated interpretation of wireline logs and lithology of two selected wells (KF-1 and KF-2) of Kribi oil field within the southeastern offshore Douala/Kribi Campo Basin was carried out for sequence stratigraphic analysis of sediments penetrated by the wells. Methods: The stratigraphic units within the wells were subdivided into depositional sequences using characteristic well log patterns that were deposited between Tertiary Miocene to lower Cretaceous. Results: Nine (9) and eight (8) depositional sequences were identified respectively for KF-1 and KF-2. The sequences comprise LST (progradational packages), TSTs (retrogradational packages) and HSTs (aggradational packages), which reflect depositional systems deposited during different phases of base-level changes. The (LST) consists of Basin Floor Fans (BFF), Slope Fans and Channel Sands deposited when sea level was low and accommodation space lower than rate of sediment influx. TST consists of retrogradational marine shales deposited during high relative sea levels and when accommodation space was higher than rate of sediment influx. HST consisted of shoreface sands displaying mostly aggradational to progradational stacking patterns. Conclusion: The rapid facies changes between successive systems tracts provide potential stratigraphic traps. Reservoir stratification and continuity vary greatly between systems tracts and this enhanced development of stratigraphic traps in the area. Basin floor fans comprise sandstone of good reservoir quality, thus huge accumulation of HC can be trapped in this reservoirs.

Keywords: Douala-Kribi-Campo Basin, reservoirs, sequence strastigraphyy, system tracks

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Authors: Sayed Ali Garossi, Azar Heidarizadi, Shahla Mohammad Ganji

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Context: Colorectal cancer is the second type of cancer-related mortality globally. Expression of cas8 (caspase 8) is closely connected to growth and metastasis of colorectal cancer.Cas8/Rip1 plays a vital role in the apoptosis pathway and resistance to chemotherapy. The aim of the present study is to investigate the pattern of gene expression in colorectal cancer and compare the differences using Real-Time PCR to find a potential biomarker candidate for colorectal cancer. Methodology: This study conducted real-time PCR to evaluate gene expression of Cas8 in colorectal cancer patients. The gene-specific primer sequences exon–exon junction was designed by OLIGO7 software for the expression of the gene under investigation. Forty-six patient samples without any chemotherapy were selected, including tumoral tissue and adjacent normal tissue samples. The age of the patients was 50 and the size of the tumors was 5.5 cm. The categories were before and after age 50. Findings: Here, we found that Caspase 8 was overexpressed in CRC tissues compared to corresponding adjacent colon tissues (Cas8: 5.2 vs. 1 ratio); high expression of Cas8 was associated with poor overall survival and independent risk factors for the prognosis of CRC patients. Conclusion: In conclusion, our study pioneered the reporting of high Casp8 expression as a predictor of poor prognosis and chemical resistance in CRC patients.Cas8 overexpression suppressed Cas 8 / Rip1-dependent apoptosis and activated the proliferation of tumor cells by activating necroptosis. The necroptosis pathway has also emerged as a new approach to anti-tumor in cancer treatment.

Keywords: Cas8, necroptosis, apoptosis, Real-Time PCR

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Authors: Lemar Blake, Chris Oura, Ayanna C. N. Phillips Savage

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Improved DNA sequencing technology has greatly enhanced bacterial identification, especially for organisms that are difficult to culture. Mycobacteriosis with consistent hyphema, bilateral exophthalmia, open mouth gape and ocular lesions, were observed in various fish populations at the School of Veterinary Medicine, Aquaculture/Aquatic Animal Health Unit. Objective: To identify the species of Mycobacterium that is affecting aquarium fish at the School of Veterinary Medicine, Aquaculture/Aquatic Animal Health Unit. Method: A total of 13 fish samples were collected and analyzed via: Ziehl-Neelsen, conventional polymerase chain reaction (PCR) and real-time PCR. These tests were carried out simultaneously for confirmation. The following combination of conventional primers: 16s rRNA (564 bp), rpoB (396 bp), sod (408 bp) were used. Concatenation of the gene fragments was carried out to phylogenetically classify the organism. Results: Acid fast non-branching bacilli were detected in all samples from homogenized internal organs. All 13 acid fast samples were positive for Mycobacterium via real-time PCR. Partial gene sequences using all three primer sets were obtained from two samples and demonstrated a novel strain. A strain 99% related to Mycobacterium marinum was also confirmed in one sample, using 16srRNA and rpoB genes. The two novel strains were clustered with the rapid growers and strains that are known to affect humans. Conclusions: Phylogenetic analysis demonstrated two novel Mycobacterium strains with the potential of being zoonotic and one strain 99% related to Mycobacterium marinum.

Keywords: polymerase chain reaction, phylogenetic, DNA sequencing, zoonotic

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Authors: Hamidreza Saberkari, Mousa Shamsi, Hossein Ahmadi, Saeed Vaali, , MohammadHossein Sedaaghi

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In the recent years, using signal processing tools for accurate identification of the protein coding regions has become a challenge in bioinformatics. Most of the genomic signal processing methods is based on the period-3 characteristics of the nucleoids in DNA strands and consequently, spectral analysis is applied to the numerical sequences of DNA to find the location of periodical components. In this paper, a novel ensemble algorithm for gene selection in DNA sequences has been presented which is based on the combination of Goertzel algorithm and anti-notch filter (ANF). The proposed algorithm has many advantages when compared to other conventional methods. Firstly, it leads to identify the coding protein regions more accurate due to using the Goertzel algorithm which is tuned at the desired frequency. Secondly, faster detection time is achieved. The proposed algorithm is applied on several genes, including genes available in databases BG570 and HMR195 and their results are compared to other methods based on the nucleotide level evaluation criteria. Implementation results show the excellent performance of the proposed algorithm in identifying protein coding regions, specifically in identification of small-scale gene areas.

Keywords: protein coding regions, period-3, anti-notch filter, Goertzel algorithm

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Authors: Kamal, Adil Ahmad

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Earthquakes are among the most versatile natural and dynamic processes, and hence a fractal model is considered to be the best representative of the same. We present a novel method to process and analyse information hidden in earthquake sequences using Fractal Dimensions and Iterative Function Systems (IFS). Spatial and temporal variations in the fractal dimensions of seismicity observed around the Indian peninsula in last 30 years are studied. This was used as a possible precursor before large earthquakes in the region. IFS images for observed seismicity in the Himalayan belt were also obtained. We scan the whole data set and coarse grain of a selected window to reduce it to four bins. A critical analysis of four-cornered chaos-game clearly shows that the spatial variation in earthquake occurrences in Himalayan range is not random. Two subzones of Himalaya have a tendency to follow each other in time.

Keywords: earthquakes, fractals, Himalaya, iterated function systems

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Authors: Manju Kanu, Subrata Sinha, Surabhi Johari

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Viral proteins of Ebola viruses belong to one of the best studied viruses; however no effective prevention against EBOV has been developed. Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with Ebola virus as a model system. Hence great challenge in the field of ebola virus research is to design universal vaccine. A combination of publicly available bioinformatics algorithms and computational tools are used to screen and select antigen sequences as potential T-cell epitopes of supertypes Human Leukocyte Antigen (HLA) alleles. MUSCLE and MOTIF tools were used to find out most conserved peptide sequences of viral proteins. Immunoinformatics tools were used for prediction of immunogenic peptides of viral proteins in zaire strains of Ebola virus. Putative epitopes for viral proteins (VP) were predicted from conserved peptide sequences of VP. Three tools NetCTL 1.2, BIMAS and Syfpeithi were used to predict the Class I putative epitopes while three tools, ProPred, IEDB-SMM-align and NetMHCII 2.2 were used to predict the Class II putative epitopes. B cell epitopes were predicted by BCPREDS 1.0. Immunogenic peptides were identified and selected manually by putative epitopes predicted from online tools individually for both MHC classes. Finally sequences of predicted peptides for both MHC classes were looked for common region which was selected as common immunogenic peptide. The immunogenic peptides were found for viral proteins of Ebola virus: epitopes FLESGAVKY, SSLAKHGEY. These predicted peptides could be promising candidates to be used as target for vaccine design.

Keywords: epitope, b cell, immunogenicity, ebola

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Authors: Khalid Alhudaib

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Potato is considered as one of the most important and potential vegetable crops in Saudi Arabia. Alfalfa mosaic virus (AMV), genus Alfamovirus, family Bromoviridae is among the broad spread of viruses in potato. During spring and fall growing seasons of potato in 2015 and 2016, several field visits were conducted in the four major growing areas of potato cultivation (Riyadh-Qaseem-Hail-Hard). The presence of AMV was detected in samples using ELISA, dot blot hybridization and/or RT-PCR. The highest occurrence of AMV was observed as 18.6% in Qaseem followed by Riyadh with 15.2% while; the lowest infection rates were recorded in Hard and Hail, 8.3 and 10.4%, respectively. The sequences of seven isolates of AMV obtained in this study were determined and the sequences were aligned with the other sequences available in the GenBank database. Analyses confirmed the low variability among AMV isolated in this study, which means that all AMV isolates may originate from the same source. Due to high incidence of AMV, other economic susceptible crops may become affected by high incidence of this virus in potato crops. This requires accurate examination of potato seed tubers to prevent the spread of the virus in Saudi Arabia. The obtained results indicated that the hybridization and ELISA are suitable techniques in the routine detection of AMV in a large number of samples while RT-PCR is more sensitive and essential for molecular characterization of AMV.

Keywords: Alfamovirus, AMV, Alfalfa mosaic virus, PCR, potato

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Authors: Jaco Oosthuizen, Nerina C. Van Der Merwe

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The National Health Laboratory Services in Bloemfontien has been the diagnostic testing facility for 1830 patients for familial breast cancer since 1997. From the cohort, 540 were comprehensively screened using High-Resolution Melting Analysis or Next Generation Sequencing for the presence of point mutations and/or indels. Approximately 90% of these patients stil remain undiagnosed as they are BRCA1/2 negative. Multiplex ligation-dependent probe amplification was initially added to screen for copy number variation detection, but with the introduction of next generation sequencing in 2017, was substituted and is currently used as a confirmation assay. The aim was to investigate the viability of utilizing internationally designed copy number variation detection assays based on mostly European/Caucasian genomic data for use within a South African context. The multiplex ligation-dependent probe amplification technique is based on the hybridization and subsequent ligation of multiple probes to a targeted exon. The ligated probes are amplified using conventional polymerase chain reaction, followed by fragment analysis by means of capillary electrophoresis. The experimental design of the assay was performed according to the guidelines of MRC-Holland. For BRCA1 (P002-D1) and BRCA2 (P045-B3), both multiplex assays were validated, and results were confirmed using a secondary probe set for each gene. The next generation sequencing technique is based on target amplification via multiplex polymerase chain reaction, where after the amplicons are sequenced parallel on a semiconductor chip. Amplified read counts are visualized as relative copy numbers to determine the median of the absolute values of all pairwise differences. Various experimental parameters such as DNA quality, quantity, and signal intensity or read depth were verified using positive and negative patients previously tested internationally. DNA quality and quantity proved to be the critical factors during the verification of both assays. The quantity influenced the relative copy number frequency directly whereas the quality of the DNA and its salt concentration influenced denaturation consistency in both assays. Multiplex ligation-dependent probe amplification produced false positives due to ligation failure when ligation was inhibited due to a variant present within the ligation site. Next generation sequencing produced false positives due to read dropout when primer sequences did not meet optimal multiplex binding kinetics due to population variants in the primer binding site. The analytical sensitivity and specificity for the South African population have been proven. Verification resulted in repeatable reactions with regards to the detection of relative copy number differences. Both multiplex ligation-dependent probe amplification and next generation sequencing multiplex panels need to be optimized to accommodate South African polymorphisms present within the genetically diverse ethnic groups to reduce the false copy number variation positive rate and increase performance efficiency.

Keywords: familial breast cancer, multiplex ligation-dependent probe amplification, next generation sequencing, South Africa

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Authors: Alisha Khanal, Gokhan Saygili

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It is commonly observed that aftershocks follow the mainshock. Aftershocks continue over a period of time with a decreasing frequency and typically there is not sufficient time for repair and retrofit between a mainshock–aftershock sequence. Usually, aftershocks are smaller in magnitude; however, aftershock ground motion characteristics such as the intensity and duration can be greater than the mainshock due to the changes in the earthquake mechanism and location with respect to the site. The seismic performance of slopes is typically evaluated based on the sliding displacement predicted to occur along a critical sliding surface. Various empirical models are available that predict sliding displacement as a function of seismic loading parameters, ground motion parameters, and site parameters but these models do not include the aftershocks. The seismic risks associated with the post-mainshock slopes ('damaged slopes') subjected to aftershocks is significant. This paper extends the empirical sliding displacement models for flexible slopes subjected to earthquake mainshock-aftershock sequences (a multi hazard approach). A dataset was developed using 144 pairs of as-recorded mainshock-aftershock sequences using the Pacific Earthquake Engineering Research Center (PEER) database. The results reveal that the combination of mainshock and aftershock increases the seismic demand on slopes relative to the mainshock alone; thus, seismic risks are underestimated if aftershocks are neglected.

Keywords: seismic slope stability, mainshock, aftershock, landslide, earthquake, flexible slopes

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Authors: Abdesselem Dakhli, Wajdi Bellil, Chokri Ben Amar

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DNA Barcode, a short mitochondrial DNA fragment, made up of three subunits; a phosphate group, sugar and nucleic bases (A, T, C, and G). They provide good sources of information needed to classify living species. Such intuition has been confirmed by many experimental results. Species classification with DNA Barcode sequences has been studied by several researchers. The classification problem assigns unknown species to known ones by analyzing their Barcode. This task has to be supported with reliable methods and algorithms. To analyze species regions or entire genomes, it becomes necessary to use similarity sequence methods. A large set of sequences can be simultaneously compared using Multiple Sequence Alignment which is known to be NP-complete. To make this type of analysis feasible, heuristics, like progressive alignment, have been developed. Another tool for similarity search against a database of sequences is BLAST, which outputs shorter regions of high similarity between a query sequence and matched sequences in the database. However, all these methods are still computationally very expensive and require significant computational infrastructure. Our goal is to build predictive models that are highly accurate and interpretable. This method permits to avoid the complex problem of form and structure in different classes of organisms. On empirical data and their classification performances are compared with other methods. Our system consists of three phases. The first is called transformation, which is composed of three steps; Electron-Ion Interaction Pseudopotential (EIIP) for the codification of DNA Barcodes, Fourier Transform and Power Spectrum Signal Processing. The second is called approximation, which is empowered by the use of Multi Llibrary Wavelet Neural Networks (MLWNN).The third is called the classification of DNA Barcodes, which is realized by applying the algorithm of hierarchical classification.

Keywords: DNA barcode, electron-ion interaction pseudopotential, Multi Library Wavelet Neural Networks (MLWNN)

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Authors: Annet Namusoke, Annet Namayanja, Peter Wasswa, Shakirah Nampijja

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Common bean cultivar G2333 which offers broad resistance for anthracnose has been widely used as a source of resistance in breeding for anthracnose resistance. The cultivar is pyramided with three genes namely CO-4, CO-5 and CO-7 and of these three genes, the CO-4 gene has been found to offer the broadest resistance. The main aim of this work was to identify and validate easily assayable PCR based co-dominant molecular markers for selection of the CO-4 gene in segregating populations derived from crosses of G2333 with RWR 1946 and RWR 2075, two commercial Andean cultivars highly susceptible to anthracnose. Marker sequences for the study were obtained by blasting the sequence of the COK-4 gene in the Phaseolus gene database. Primer sequence pairs that were not provided from the Phaseolus gene database were designed by the use of Primer3 software. PCR conditions were optimized and the PCR products were run on 6% HPAGE gel. Results of the polymorphism test indicated that out of 18 identified markers, only two markers namely BM588 and BM211 behaved co-dominantly. Phenotypic evaluation for reaction to anthracnose disease was done by inoculating 21days old seedlings of three parents, F1 and F2 populations with race 7 of Colletotrichum lindemuthianum in the humid chamber. DNA testing of the BM588 marker onto the F2 segregating population of the crosses RWR 1946 x G 2333 and RWR 2075 x G2333 further revealed that the marker BM588 co-segregated with disease resistance with co-dominance of two alleles of 200bp and 400bp, fitting the expected segregation ratio of 1:2:1. The BM588 marker was significantly associated with disease resistance and gave promising results for marker assisted selection of the CO-4 gene in the breeding lines. Activities to validate the BM211 marker are also underway.

Keywords: codominant, Colletotrichum lindemuthianum, MAS, Phaseolus vulgaris

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Authors: Makhosi Buthelezi

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Mitochondrial DNA cytochrome c oxidase I (COI) gene analyses linked the South African groundnut leaf miner (GLM) to the Australian soya bean moth Aproaerema simplexella (Walker) and Indian Aproaerema modicella (Deventer). Thus, the genetic relatedness of GLM, A. simplexela, and A. modicella was examined by performing mitochondrial and nuclear (COI, cytochrome oxidase subunit II (COII), mitochondrial cytochrome b (CYTB), nuclear ribosomal 28S (28S) and intergenic spacer elongation factor-1 alpha ( EF-1 ALPHA) on 44 specimens collected from South Africa, four from Mozambique, and three each from single locations in India and Australia. Phylogenetic analyses were conducted using the Maximum Parsimony (MP) and Neighbour-Joining (NJ) methods. All of the datasets of the five DNA gene regions that were sequenced were also analyzed using the Basic Local Alignment Search Tool (BLAST) to find the closest matches for inclusion in the phylogenetic trees as outgroups and for purposes of information. In the phylogenetic trees for COI, COII, cytb and EF-1 ALPHA, a similar pattern was observed in the way that the sequences assembled into different groups; i.e., some sequences of A. simplexella from Australia were grouped separately from the others, but some Australian sequences grouped with those of the GLM from South Africa, India, and Mozambique. In the phylogenetic tree for 28S, all sequences from South Africa, Australia, India, and Mozambique grouped together and formed one group. For COI, genetic pairwise distance ranged from 0.97 to 3.60 %, for COII it ranged from 0.19% to 2.32%, for cytb it ranged from 0.25 to 9.77% and for EF-1 ALPHA it ranged 0.48 to 6.99%. Results of this study indicate that these populations are genetically related and presumably constitute a single species. Thus, further molecular and morphological studies need to be undertaken in order to resolve this apparent conundrum on the taxonomy of these populations.

Keywords: aproaerema modicella, aproaerema simplexella, mitochondrial DNA, nuclear DNA

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Authors: Yingxu Wang

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This work presents basic research on the recursive structures and dual sequential patterns of primes for the formal proof of the Twin-Prime Conjecture (TPC). A rigorous methodology of Twin-Prime Decomposition (TPD) is developed in MATLAB to inductively verify potential twins in the dual sequences of primes. The key finding of this basic study confirms that the dual sequences of twin primes are not only symmetric but also infinitive in the unique base 6 cycle, except a limited subset of potential pairs is eliminated by the lack of dual primality. Both theory and experiments have formally proven that the infinity of twin primes stated in TPC holds in the P x P space.

Keywords: number theory, primes, twin-prime conjecture, dual primes (P x P), twin prime decomposition, formal proof, algorithm

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Authors: Seung Min Kim, Lyu Jin Jun, Joon Bum Jeong

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The Myxosporea to cause emaciation disease in the olive flounder (Paralichthys olivaceus) is a pathogen to cause severe losses in the aquafarming industry in Korea. The 3,362 bp of DNA nucleotide sequences of four myxosporean strains (EM-HM-12, EM-MA-13, EM-JJ-14, and EM-MS-15) detected by PCR method from olive flounder suffering from emaciation disease in Korea during 2012-2015 were sequenced and deposited in GenBank database (GenBank accession numbers: KU377574, KT321705, KU377575 and KU377573, respectively). The homologies of DNA nucleotide sequences of four strains were compared to each other and were more than 99.7% homologous between the four strains. All of the strains were identified as Parvicapsula petunia based on the results of phylogenetic analysis. The results in this study would be useful for the research of emaciation disease in olive flounder of Korea.

Keywords: disease, emaciation, olive flounder, phylogenetic analysis

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Authors: Garadi Ahmed, Boubakar S. Bouazza

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The Zero Cross-Correlation (C, w) code is a family of binary sequences of length C and constant Hamming-weight, the cross correlation between any two sequences equal zero. In this paper, we evaluate the performance of ZCC code based on Balanced Incomplete Block Design (BIBD) for Spectral Amplitude Coding Optical Code Division Multiple Access (SAC-OCDMA) system using direct detection. The BER obtained is better than 10-9 for five simultaneous users.

Keywords: spectral amplitude coding-optical code-division-multiple-access (SAC-OCDMA), phase induced intensity noise (PIIN), balanced incomplete block design (BIBD), zero cross-correlation (ZCC)

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Authors: V. Karuppaiah, J. C. Padaria, C. Srivastava

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The tobacco caterpillar, Spodoptera litura Fab (Lepidoptera: Noctuidae) is a polyphagous pest various field and horticulture crops in India. Pest had virtually developed resistance to all commonly used insecticides. Enhanced detoxification is the prime mechanism that is dictated by detoxification different enzymes and carboxylesterase is one of the major enzyme responsible development of resistance. In India, insecticide resistance studies on S. litura are mainly deployed on detoxification enzymes activity and investigation at gene level alteration i.e. at nucleotide level is very merger. In the present study, we collected the S. litura larvae from three different cauliflower growing belt viz., IARI, New Delhi (Delhi), Palari, Sonepat (Haryana) and Varanasi (Uttar Pradesh) to study the role of carboxylesterase activity and its gene level variation The CarE activity was measured using UV-VIS spectrophotometer with 3rd instar larvae of S. litura. The elevated activity of CarE was observed in Sonepat strain (28.09 ± 0.09 µmol/min/mg of protein) followed by Delhi (26.72 ± 0.04 µmol/min/mg of protein) and Varanasi strain (10.00 ± 0.44 µmol/min/mg of protein) of S. litura. The genomic DNA was isolated from 3rd instar larvae and CarE gene was amplified using a primer sequence, F:5’tccagagttccttgtcaggcac3’; R:5’ctgcatcaagcatgtctc3. CarE gene, about 500bp was partially amplified, sequenced and submitted to NCBI (Accession No. KF835886, KF835887 and KF835888). The sequence data revealed polymorphism at nucleotide level in all the three strains and gene found to have 88 to 97% similarity with previous available nucleotide sequences of S. litura, S. littoralis and S. exiqua. The polymorphism at the nucleotide level could be a reason for differential activity of carboxylesterase enzymes among the strains. However, investigation at gene expression level would be useful to analyze the overproduction of carboxylesterase enzyme.

Keywords: carboxylesterase, CarE gene, nucleotide polymorphism, insecticide resistance, spodoptera litura

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Authors: Jorge Maestre Vidal, Ana Lucila Sandoval Orozco, Luis Javier García Villalba

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With the increase in popularity of mobile devices, new and varied forms of malware have emerged. Consequently, the organizations for cyberdefense have echoed the need to deploy more effective defensive schemes adapted to the challenges posed by these recent monitoring environments. In order to contribute to their development, this paper presents a malware detection strategy for mobile devices based on sequence alignment algorithms. Unlike the previous proposals, only the system calls performed during the startup of applications are studied. In this way, it is possible to efficiently study in depth, the sequences of system calls executed by the applications just downloaded from app stores, and initialize them in a secure and isolated environment. As demonstrated in the performed experimentation, most of the analyzed malicious activities were successfully identified in their boot processes.

Keywords: android, information security, intrusion detection systems, malware, mobile devices

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Authors: Jingjie Li, Wenjie Hu

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Phraseological units in academic English texts have been a central focus in recent corpus linguistic research. A wide variety of phraseological units have been explored, including collocations, chunks, lexical bundles, patterns, semantic sequences, etc. This paper describes a special category of clause-level phraseological units, namely, Characteristic Sentence Stems (CSSs), with a view to describing their defining criteria and extraction method. CSSs are contiguous lexico-grammatical sequences which contain a subject-predicate structure and which are frame expressions characteristic of academic writing. The extraction of CSSs consists of six steps: Part-of-speech tagging, n-gram segmentation, structure identification, significance of occurrence calculation, text range calculation, and overlapping sequence reduction. Significance of occurrence calculation is the crux of this study. It includes the computing of both the internal association and the boundary independence of a CSS and tests the occurring significance of the CSS from both inside and outside perspectives. A new normalization algorithm is also introduced into the calculation of LocalMaxs for reducing overlapping sequences. It is argued that many sentence stems are so recurrent in academic texts that the most typical of them have become the habitual ways of making meaning in academic writing. Therefore, studies of CSSs could have potential implications and reference value for academic discourse analysis, English for Academic Purposes (EAP) teaching and writing.

Keywords: characteristic sentence stem, extraction method, phraseological unit, the statistical measure

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Authors: Othman Elmahdy Othman, Agnés Germot, Daniel Petit, Muhammad Khodary, Abderrahman Maftah

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Recently, the control (D-loop) and cytochrome b (Cyt b) regions of mtDNA have received more attention due to their role in the genetic diversity and phylogenetic studies in different livestock which give important knowledge towards the genetic resource conservation. Studies based on sequencing of sheep mitochondrial DNA showed that there are five maternal lineages in the world for domestic sheep breeds; A, B, C, D and E. By using cytochrome B sequencing, we aimed to clarify the genetic affinities and phylogeny of six Egyptian sheep breeds. Blood samples were collected from 111 animals belonging to six Egyptian sheep breeds; Barki, Rahmani, Ossimi, Saidi, Sohagi and Fallahi. The total DNA was extracted and the specific primers were used for conventional PCR amplification of the cytochrome B region of mtDNA. PCR amplified products were purified and sequenced. The alignment of sequences was done using BioEdit software and DnaSP 5.00 software was used to identify the sequence variation and polymorphic sites in the aligned sequences. The result showed that the presence of 39 polymorphic sites leading to the formation of 29 haplotypes. The haplotype diversity in six tested breeds ranged from 0.643 in Rahmani breed to 0.871 in Barki breed. The lowest genetic distance was observed between Rahmani and Saidi (D: 1.436 and Dxy: 0.00127) while the highest distance was observed between Ossimi and Sohagi (D: 6.050 and Dxy: 0.00534). Neighbour-joining (Phylogeny) tree was constructed using Mega 5.0 software. The sequences of 111 analyzed samples were aligned with references sequences of different haplogroups; A, B, C, D and E. The phylogeny result showed the presence of four haplogroups; HapA, HapB, HapC and HapE in the examined samples whereas the haplogroup D was not found. The result showed that 88 out of 111 tested animals cluster with haplogroup B (79.28%), whereas 12 tested animals cluster with haplogroup A (10.81%), 10 animals cluster with haplogroup C (9.01%) and one animal belongs to haplogroup E (0.90%).

Keywords: phylogeny, genetic biodiversity, MtDNA, cytochrome B, Egyptian sheep

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Authors: Mi-Ju Kim, Hae-Yeong Kim

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Product mislabeling and adulteration have been increasing the concerns in processed meat products. Relatively inexpensive pork meat compared to meat such as beef was adulterated for economic benefit. These food fraud incidents related to pork were concerned due to economic, religious and health reasons. In this study, a rapid on-site detection method using loop-mediated isothermal amplification (LAMP) was developed for the simultaneous identification of beef and pork. Each specific LAMP primer for beef and pork was designed targeting on mitochondrial D-loop region. The LAMP assay reaction was performed at 65 ℃ for 40 min. The specificity of each primer for beef and pork was evaluated using DNAs extracted from 13 animal species including beef and pork. The sensitivity of duplex LAMP assay was examined by serial dilution of beef and pork DNAs, and reference binary mixtures. This assay was applied to processed meat products including beef and pork meat for monitoring. Each set of primers amplified only the targeted species with no cross-reactivity with animal species. The limit of detection of duplex real-time LAMP was 1 pg for each DNA of beef and pork and 1% pork in a beef-meat mixture. Commercial meat products that declared the presence of beef and/or pork meat on the label showed positive results for those species. This method was successfully applied to detect simultaneous beef and pork meats in processed meat products. The optimized duplex LAMP assay can identify simultaneously beef and pork meat within less than 40 min. A portable real-time fluorescence device used in this study is applicable for on-site detection of beef and pork in processed meat products. Thus, this developed assay was considered to be an efficient tool for monitoring meat products.

Keywords: beef, duplex real-time LAMP, meat identification, pork

Procedia PDF Downloads 225

Authors: Shahid Hussain Abro, Siamak Zohari, Lena H. M. Renström, Désirée S. Jansson, Faruk Otman, Karin Ullman, Claudia Baule

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Infectious bronchitis is an acute, highly contagious respiratory, nephropathogenic and reproductive disease of poultry that is caused by infectious bronchitis virus (IBV). The present study used a large data set of structural gene sequences, including newly generated ones and sequences available in the GenBank database to further analyze the diversity and to identify selective pressures and recombination spots. There were some deletions or insertions in the analyzed regions in isolates of the Italy-02 and D274 genotypes. Whereas, there were no insertions or deletions observed in the isolates of the Massachusetts and 4/91 genotype. The hypervariable nucleotide sequence regions spanned positions 152–239, 554–582, 686–737 and 802–912 in the S1 sub-unit of the all analyzed genotypes. The nucleotide sequence data of the E gene showed that this gene was comparatively unstable and subjected to a high frequency of mutations. The M gene showed substitutions consistently distributed except for a region between nucleotide positions 250–680 that remained conserved. The lowest variation in the nucleotide sequences of ORF5a was observed in the isolates of the D274 genotype. While, ORF5b and N gene sequences showed highly conserved regions and were less subjected to variation. Genes ORF3a, ORF3b, M, ORF5a, ORF5b and N presented negative selective pressure among the analyzed isolates. However, some regions of the ORFs showed favorable selective pressure(s). The S1 and E proteins were subjected to a high rate of mutational substitutions and non-synonymous amino acids. Strong signals of recombination breakpoints and ending break point were observed in the S and N genes. Overall, the results of this study revealed that very likely the strong selective pressures in E, M and the high frequency of substitutions in the S gene can probably be considered the main determinants in the evolution of IBV.

Keywords: IBV, avian infectious bronchitis, structural genes, genotypes, genetic diversity

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Authors: Houxiang Zhu, Chun Liang

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The CRISPR-Cpf1 system has been successfully applied in genome editing. However, target efficiency of the CRISPR-Cpf1 system varies among different gRNA sequences. The published CRISPR-Cpf1 gRNA data was reanalyzed. Many sequences and structural features of gRNAs (e.g., the position-specific nucleotide composition, position-nonspecific nucleotide composition, GC content, minimum free energy, and melting temperature) correlated with target efficiency were found. Using machine learning technology, a support vector machine (SVM) model was created to predict target efficiency for any given gRNAs. The first web service application, CRISPR-DT (CRISPR DNA Targeting), has been developed to help users design optimal gRNAs for the CRISPR-Cpf1 system by considering both target efficiency and specificity. CRISPR-DT will empower researchers in genome editing.

Keywords: CRISPR-Cpf1, genome editing, target efficiency, target specificity

Procedia PDF Downloads 264