Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 3186

Search results for: polymerase chain reaction

3186 The Influence of the Moving Speeds of DNA Droplet on Polymerase Chain Reaction

Authors: Jyh Jyh Chen, Fu H. Yang, Chen W. Wang, Yu M. Lin

Abstract:

In this work, a reaction chamber is reciprocated among three temperature regions by using an oscillatory thermal cycling machine. Three cartridge heaters are collocated to heat three aluminum blocks in order to achieve PCR requirements in the reaction chamber. The effects of various chamber moving speeds among different temperature regions on the chamber temperature profiles are presented. To solve the evaporation effect of the sample in the PCR experiment, the mineral oil and the cover lid are used. The influences of various extension times on DNA amplification are also demonstrated. The target fragments of the amplification are 385-bp and 420-bp. The results show when the forward speed is set at 6 mm/s and the backward speed is 2.4 mm/s, the temperature required for the experiment can be achieved. It is successful to perform the amplification of DNA fragments in our device.

Keywords: oscillatory, polymerase chain reaction, reaction chamber, thermal cycling machine

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3185 Comparison of Sensitivity and Specificity of Pap Smear and Polymerase Chain Reaction Methods for Detection of Human Papillomavirus: A Review of Literature

Authors: M. Malekian, M. E. Heydari, M. Irani Estyar

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Human papillomavirus (HPV) is one of the most common sexually transmitted infection, which may lead to cervical cancer as the main cause of it. With early diagnosis and treatment in health care services, cervical cancer and its complications are considered to be preventable. This study was aimed to compare the efficiency, sensitivity, and specificity of Pap smear and polymerase chain reaction (PCR) in detecting HPV. A literature search was performed in Google Scholar, PubMed and SID databases using the keywords 'human papillomavirus', 'pap smear' and 'polymerase change reaction' to identify studies comparing Pap smear and PCR methods for the detection. No restrictions were considered.10 studies were included in this review. All samples that were positive by pop smear were also positive by PCR. However, there were positive samples detected by PCR which was negative by pop smear and in all studies, many positive samples were missed by pop smear technique. Although The Pap smear had high specificity, PCR based HPV detection was more sensitive method and had the highest sensitivity. In order to promote the quality of detection and high achievement of the maximum results, PCR diagnostic methods in addition to the Pap smear are needed and Pap smear method should be combined with PCR techniques according to the high error rate of Pap smear in detection.

Keywords: human papillomavirus, cervical cancer, pap smear, polymerase chain reaction

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3184 PTFE Capillary-Based DNA Amplification within an Oscillatory Thermal Cycling Device

Authors: Jyh J. Chen, Fu H. Yang, Ming H. Liao

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This study describes a capillary-based device integrated with the heating and cooling modules for polymerase chain reaction (PCR). The device consists of the reaction polytetrafluoroethylene (PTFE) capillary, the aluminum blocks, and is equipped with two cartridge heaters, a thermoelectric (TE) cooler, a fan, and some thermocouples for temperature control. The cartridge heaters are placed into the heating blocks and maintained at two different temperatures to achieve the denaturation and the extension step. Some thermocouples inserted into the capillary are used to obtain the transient temperature profiles of the reaction sample during thermal cycles. A 483-bp DNA template is amplified successfully in the designed system and the traditional thermal cycler. This work should be interesting to persons involved in the high-temperature based reactions and genomics or cell analysis.

Keywords: polymerase chain reaction, thermal cycles, capillary, TE cooler

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3183 Detection of Respiratory Syncytial Virus (hRSV) by PCR Technique in Lower Respiratory Tract Infection (LRTI) in Babylon City

Authors: Amal Raqib Shameran, Ghanim Aboud Al-Mola

Abstract:

Respiratory syncytial virus (hRSV) is the major pathogens of respiratory tract infections (RTI) among infants and children in the world. They are classified in family Paramyxoviridae and sub-family Pneumovirinae. The current work aimed to detect the role of RSV in the lower respiratory tract infection (LRTI) in Hilla, Iraq. The samples were collected from 50 children who were admitted to hospital suffering from lower respiratory tract infections (LRTI). 50 nasal and pharyngeal swabs were taken from patients at the period from January 2010 till April 2011, hospitalized in Hilla Maternity and Children Hospital. The results showed that the proportion of children infected with hRSV accounted for 24% 12/50 with lower respiratory tract infections (LRTI) when they tested by polymerase chain reaction (RT-PCR).

Keywords: respiratory syncytial virus, respiratory tract infections, infants, polymerase chain reaction (PCR)

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3182 Polymerase Chain Reaction Analysis and Random Amplified Polymorphic DNA of Agrobacterium Tumefaciens

Authors: Abeer M. Algeblawi

Abstract:

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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3181 Negative RT-PCR in a Newborn Infected with Zika Virus: A Case Report

Authors: Vallejo Michael, Acuña Edgar, Roa Juan David, Peñuela Rosa, Parra Alejandra, Casallas Daniela, Rodriguez Sheyla

Abstract:

Congenital Zika Virus Syndrome is an entity composed by a variety of birth defects presented in newborns that have been exposed to the Zika Virus during pregnancy. The syndrome characteristic features are severe microcephaly, cerebral tissue abnormalities, ophthalmological abnormalities such as uveitis and chorioretinitis, arthrogryposis, clubfoot deformity and muscular tone abnormalities. The confirmatory test is the Reverse transcription polymerase chain reaction (RT-PCR) associated to the physical findings. Here we present the case of a newborn with microcephaly whose mother presented a confirmed Zika Virus infection during the third trimester of pregnancy, despite of the evident findings and the history of Zika infection the RT-PCR in amniotic and cerebrospinal fluid of the newborn was negative. RT-PCR has demonstrated a low sensibility in samples with low viral loads, reason why, we propose a clinical diagnosis in patients with clinical history of Zika Virus infection during pregnancy accompanied by evident clinical manifestations of the child.

Keywords: congenital, Zika virus, microcephaly, reverse transcriptase polymerase chain reaction

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3180 The Predictive Value of Micro Rna 451 on the Outcome of Imatinib Treatment in Chronic Myeloid Leukemia Patients

Authors: Nehal Adel Khalil, Amel Foad Ketat, Fairouz Elsayed Mohamed Ali, Nahla Abdelmoneim Hamid, Hazem Farag Manaa

Abstract:

Background: Chronic myeloid leukemia (CML) represents 15% of adult leukemias. Imatinib Mesylate (IM) is the gold standard treatment for new cases of CML. Treatment with IM results in improvement of the majority of cases. However, about 25% of cases may develop resistance. Sensitive and specific early predictors of IM resistance in CML patients have not been established to date. Aim: To investigate the value of miR-451 in CML as an early predictor for IM resistance in Egyptian CML patients. Methods: The study employed Real time Polymerase Reaction (qPCR) technique to investigate the leucocytic expression of miR-451 in fifteen newly diagnosed CML patients (group I), fifteen IM responder CML patients (group II), fifteen IM resistant CML patients (group III) and fifteen healthy subjects of matched age and sex as a control group (group IV). The response to IM was defined as < 10% BCR-ABL transcript level after 3 months of therapy. The following parameters were assessed in subjects of all the studied groups: 1- Complete blood count (CBC). 2- Measurement of plasma level of miRNA 451 using real-time Polymerase Chain Reaction (qPCR). 3- Detection of BCR-ABL gene mutation in CML using qPCR. Results: The present study revealed that miR-451 was significantly down-regulated in leucocytes of newly diagnosed CML patients as compared to healthy subjects. IM responder CML patients showed an up-regulation of miR- 451 compared with IM resistant CML patients. Conclusion: According to the data from the present study, it can be concluded that leucocytic miR- 451 expression is a useful additional follow-up marker for the response to IM and a promising prognostic biomarker for CML.

Keywords: chronic myeloid leukemia, imatinib resistance, microRNA 451, Polymerase Chain Reaction

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3179 Molecular Comparison of HEV Isolates from Sewage & Humans at Western India

Authors: Nidhi S. Chandra, Veena Agrawal, Debprasad Chattopadhyay

Abstract:

Background: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in developing countries. It spreads feco orally mainly due to contamination of drinking water by sewage. There is limited data on the genotypic comparison of HEV isolates from sewage water and humans. The aim of this study was to identify genotype and conduct phylogenetic analysis of HEV isolates from sewage water and humans. Materials and Methods: 14 sewage water and 60 serum samples from acute sporadic hepatitis E cases (negative for hepatitis A, B, C) were tested for HEV-RNA by nested polymerase chain reaction (RTnPCR) using primers designed with in RdRp (RNA dependent RNA polymerase) region of open reading frame-1 (ORF-1). Sequencing was done by ABI prism 310. The sequences (343 nucleotides) were compared with each other and were aligned with previously reported HEV sequences obtained from GeneBank, using Clustal W software. A Phylogenetic tree was constructed by using PHYLIP version 3.67 software. Results: HEV-RNA was detected in 49/ 60 (81.67%) serum and 5/14 (35.71%) sewage samples. The sequences obtained from 17 serums and 2 sewage specimens belonged to genotype I with 85% similarity and clustering with previously reported human HEV sequences from India. HEV isolates from human and sewage in North West India are genetically closely related to each other. Conclusion: These finding suggest that sewage acts as reservoir of HEV. Therefore it is important that measures are taken for proper waste disposal and treatment of drinking water to prevent outbreaks and epidemics due to HEV.

Keywords: hepatitis E virus, nested polymerase chain reaction, open reading frame-1, nucleotidies

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3178 Molecular Characterization of Dirofilaria repens in Dogs from Karnataka, India

Authors: D. S. Malatesh, K. J. Ananda, C. Ansar Kamran, K. Ganesh Udupa

Abstract:

Dirofilaria repens is a mosquito-borne filarioid nematode of dogs and other carnivores and accidentally affects humans. D. repens is reported in many countries, including India. Subcutaneous dirofilariosis caused by D. repens is a zoonotic disease, widely distributed throughout Europe, Asia, and Africa, with higher prevalence reported in dogs from Sri Lanka (30-60%), Iran (61%) and Italy (21-25%). Dirofilariasis in dogs was diagnosed by detection of microfilariae in blood. Identification of different Dirofilaria species was done by using molecular methods like polymerase chain reaction (PCR). Even though many researchers reported molecular evidence of D. repens across India, to our best knowledge there is no data available on molecular diagnosis of D. repens in dogs and its zoonotic implication in Karnataka state a southern state in India. The aim of the present study was to identify the Dirofilaria species occurring in dogs from Karnataka, India. Out of 310 samples screened for the presence of microfilariae using traditional diagnostic methods, 99 (31.93%) were positive for the presence of microfilariae. Based on the morphometry, the microfilariae were identified as D. repens. For confirmation of species, the samples were subjected to PCR using pan filarial primers (DIDR-F1, DIDR-R1) for amplification of internal transcribed spacer region 2 (ITS2) of the ribosomal DNA. The PCR product of 484 base pairs on agarose gel was indicative of D. repens. Hence, a single PCR reaction using pan filarial primers can be used to differentiate filarial species found in dogs. The present study confirms that dirofilarial species occurring in dogs from Karnataka is D. repens and further sequencing studies are needed for genotypic characterization of D. repens.

Keywords: Dirofilaria repens, molecular characterization, polymerase chain reaction, Karnataka, India

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3177 Detection and Dissemination of Putative Virulence Genes from Brucella Species Isolated from Livestock in Eastern Cape Province of South Africa

Authors: Rudzani Manafe, Ezekiel Green

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Brucella, has many different virulence factors that act as a causative agent of brucellosis, depending on the environment and other factors, some factors may play a role more than others during infection and as a result, play a role in becoming a causative agent for pathogenesis. Brucella melitensis and Brucella abortus are considered to be pathogenic to humans. The genetic regularity of nine potential causes of virulence of two Brucella species in Eastern Cape livestock have been examined. A hundred and twenty isolates obtained from Molecular Pathogenesis and Molecular Epidemiology Research Group (MPMERG) were used for this study. All isolates were grown on Brucella agar medium. Nine primer pairs were used for the detection of virB2, virB5, vceC, btpA, btpB, prpA, betB, bpe275, and bspB virulence factors using Polymerase chain reaction (PCR). Approximately 100% was observed for genes BecC and BetB from B. arbotus. While the lowest gene observed was PrpA at 4.6% from B. arbotus. BetB was detected in 34.7%, while virB2 and prpA (0%) were not detected in B. melitensis. The results from this research suggest that most isolates of Brucella have virulence-related genes associated with disease pathogenesis. Finally, our findings showed that Brucella strains in the Eastern Cape Province are extremely virulent as virulence characteristics exist in most strains investigated.

Keywords: putative virulence genes, brucella, polymerase chain reaction, milk

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3176 Using Polymerase Chain Reaction Technique to Observe the Resistant Strains of Pectinophora gossypiella against Cry1Ac Expressing Cotton

Authors: Zunnu Raen Akhtar, U. Irshad, M. Majid

Abstract:

Due to the widespread cultivation of transgenic cotton, intense selection pressure resulted in resistant allele in pink bollworm, Pectinophora gossypiella (Gelechiidae: Lepidoptera). A resistant strain of pink bollworm against transgenic cotton has become a challenge to Integrated Resistance Management (IRM) in the World. Laboratory and field studies were conducted to determine the resistant strains of pink bollworm by performing bioassay, extracting the DNA, conducting PCR of both laboratory as well as field collected pink bollworms to observe the developed resistance. In all of the studies, two Bt varieties FH-142 and FH-118 expressing Cry1Ac compared to non-Bt (Control) were tested against pink bollworm. In the laboratory, bioassay results showed that there was no significant mortality difference between Bt and non-Bt varieties. Similar mortality percentage was observed in transgenic and non-transgenic (control) variety. Insects which were survived after bioassay, as well as those collected from the Bt cotton fields, were selected for further molecular studies. DNA extraction followed by PCR was conducted to check the resistant strains in pink bollworm. In field studies, we also observed the population dynamics of pink boll worms on Bt as compared to non-Bt varieties. Laboratory and field studies confirmed that resistant strains occurs in Pakistani Bt cotton fields. Different strategies should be adopted to combat that serious prevailing resistance issues.

Keywords: transgenic cotton, resistance, pectinophora gossypiella, , integrated resistance management (IRM), polymerase chain reaction (PCR)

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3175 The Prevalence of Blood-Borne Viral Infections among Autopsy Cases in Jordan

Authors: Emad Al-Abdallat, Faris G. Bakri, Azmi Mahafza, Rayyan Al Ali, Nidaa Ababneh, Ahmed Idhair

Abstract:

Background: Morgues are high-risk areas for the spread of infection from the cadavers to the staff during the postmortem examination. Infection can spread from corpses to workers by the airborne route, by direct contact, or from needle and sharp object injuries. Objective: Knowledge about the prevalence of these infections among autopsies is prudent to appreciate any risk of transmission and to further enforce safety measures. Method: A total of 242 autopsies were tested. Age ranged from 3 days to 94 years (median 75.5 years, mean 45.3 (21.9 ± SD)). There were 172 (71%) males. Results: The cause of death was considered natural in 137 (56.6%) cases, accidental in 89 (36.8%), homicidal in 9 (3.7%), suicidal in 4 (1.7%), and unknown in 3 (1.2%). Hepatitis B surface antigen was positive in 5 (2.1%) cases. Hepatitis C virus antibody was detected in 5 (2.1%) cases and the hepatitis C virus polymerase chain reaction was positive in 2 of them (0.8%). HIV antibody was not detected in any of the cases. Conclusions: Autopsies can be associated with exposure to blood borne viruses. Autopsies performed during the study period were tested for hepatitis B surface antigen, hepatitis C virus antibody, and human immunodeficiency virus antibody. Positive tests were subsequently confirmed by polymerase chain reaction. There is low prevalence of infections with these viruses in our autopsy cases. However, the risk of transmission remains a threat. Healthcare workers in the forensic departments should adhere to standard precautions.

Keywords: autopsy, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, Jordan

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3174 Identification of Babesia ovis Through Polymerase Chain Reaction in Sheep and Goat in District Muzaffargarh, Pakistan

Authors: Muhammad SAFDAR, Mehmet Ozaslan, Musarrat Abbas Khan

Abstract:

Babesiosis is a haemoparasitic disease due to the multiplication of protozoan’s parasite, Babesia ovis in the red blood cells of the host, and contributes numerous economical losses, including sheep and goat ruminants. The early identification and successful treatment of Babesia Ovis spp. belong to the key steps of control and health management of livestock resources. The objective of this study was to construct a polymerase chain reaction (PCR) based method for the detection of Babesia spp. in small ruminants and to determine the risk factors involved in the spreading of babesiosis infections. A total of 100 blood samples were collected from 50 sheep and 50 goats along with different areas of Muzaffargarh, Pakistan, from randomly selected herds. Data on the characteristics of sheep and goats were collected through questionnaires. Of 100 blood samples examined, 18 were positive for Babesia ovis upon microscopic studies, whereas 11 were positive for the presence of Babesia spp. by PCR assay. For the recognition of parasitic DNA, a set of 500bp oligonucleotide was designed by PCR amplification with sequence 18S rRNA gene for B. ovis. The prevalence of babesiosis in small ruminant’s sheep and goat detected by PCR was significantly higher in female animals (28%) than male herds (08%). PCR analysis of the reference samples showed that the detection limit of the PCR assay was 0.01%. Taken together, all data indicated that this PCR assay was a simple, fast, specific detection method for Babesia ovis species in small ruminants compared to other available methods.

Keywords: Babesia ovis, PCR amplification, 18S rRNA, sheep and goat

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3173 Identifying Pathogenic Mycobacterium Species Using Multiple Gene Phylogenetic Analysis

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

Abstract:

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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3172 HIV Incidence among Men Who Have Sex with Men Measured by Pooling Polymerase Chain Reaction, and Its Comparison with HIV Incidence Estimated by BED-Capture Enzyme-Linked Immunosorbent Assay and Observed in a Prospective Cohort

Authors: Mei Han, Jinkou Zhao, Yuan Yao, Liangui Feng, Xianbin Ding, Guohui Wu, Chao Zhou, Lin Ouyang, Rongrong Lu, Bo Zhang

Abstract:

To compare the HIV incidence estimated using BED capture enzyme linked immunosorbent assay (BED-CEIA) and observed in a cohort against the HIV incidence among men who have sex with men (MSM) measured by pooling polymerase chain reaction (pooling-PCR). A total of 617 MSM subjects were included in a respondent driven sampling survey in Chongqing in 2008. Among the 129 that were tested HIV antibody positive, 102 were defined with long-term infection, 27 were assessed for recent HIV infection (RHI) using BED-CEIA. The remaining 488 HIV negative subjects were enrolled to the prospective cohort and followed-up every 6 months to monitor HIV seroconversion. All of the 488 HIV negative specimens were assessed for acute HIV infection (AHI) using pooling-PCR. Among the 488 negative subjects in the open cohort, 214 (43.9%) were followed-up for six months, with 107 person-years of observation and 14 subjects seroconverted. The observed HIV incidence was 12.5 per 100 person-years (95% CI=9.1-15.7). Among the 488 HIV negative specimens, 5 were identified with acute HIV infection using pooling-PCR at an annual rate of 14.02% (95% CI=1.73-26.30). The estimated HIV-1 incidence was 12.02% (95% CI=7.49-16.56) based on BED-CEIA. The HIV incidence estimated with three different approaches was different among subgroups. In the highly HIV prevalent MSM, it costs US$ 1724 to detect one AHI case, while detection of one case of RHI with BED assay costs only US$ 42. Three approaches generated comparable and high HIV incidences, pooling PCR and prospective cohort are more close to the true level of incidence, while BED-CEIA seemed to be the most convenient and economical approach for at-risk population’s HIV incidence evaluation at the beginning of HIV pandemic. HIV-1 incidences were alarmingly high among MSM population in Chongqing, particularly within the subgroup under 25 years of age and those migrants aged between 25 to 34 years.

Keywords: BED-CEIA, HIV, incidence, pooled PCR, prospective cohort

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3171 Frequency of BCR-ABL Fusion Transcript Types with Chronic Myeloid Leukemia by Multiplex Polymerase Chain Reaction in Srinagarind Hospital, Khon Kaen Thailand

Authors: Kanokon Chaicom, Chitima Sirijerachai, Kanchana Chansung, Pinsuda Klangsang, Boonpeng Palaeng, Prajuab Chaimanee, Pimjai Ananta

Abstract:

Chronic myeloid leukemia (CML) is characterized by the consistent involvement of the Philadelphia chromosome (Ph), which is derived from a reciprocal translocation between chromosome 9 and 22, the main product of the t(9;22) (q34;q11) translocation, is found in the leukemic clone of at least 95% of CML patients. There are two major forms of the BCR/ABL fusion gene, involving ABL exon 2, but including different exons of BCR gene. The transcripts b2a2 (e13a2) or b3a2 (e14a2) code for a p210 protein. Another fusion gene leads to the expression of an e1a2 transcript, which codes for a p190 protein. Other less common fusion genes are b3a3 or b2a3, which codes for a p203 protein and e19a2 (c3a2) transcript, which codes for a p230 protein. Its frequency varies in different populations. In this study, we aimed to report the frequency of BCR-ABL fusion transcript types with CML by multiplex PCR (polymerase chain reaction) in Srinagarind Hospital, Khon Kaen, Thailand. Multiplex PCR for BCR-ABL was performed on 58 patients, to detect different types of BCR-ABL transcripts of the t (9; 22). All patients examined were positive for some type of BCR/ABL rearrangement. The majority of the patients (93.10%) expressed one of the p210 BCR-ABL transcripts, b3a2 and b2a2 transcripts were detected in 53.45% and 39.65% respectively. The expression of an e1a2 transcript showed 3.75%. Co-expression of p210/p230 was detected in 3.45%. Co-expression of p210/p190 was not detected. Multiplex PCR is useful, saves time and reliable in the detection of BCR-ABL transcript types. The frequency of one or other rearrangement in CML varies in different population.

Keywords: chronic myeloid leukemia, BCR-ABL fusion transcript types, multiplex PCR, frequency of BCR-ABL fusion

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3170 In vitro and vivo Studies for Assessing the Anti-Proliferative, Anti-Migration and Apoptotic Activity of A. squamosa L. Leaves Extract

Authors: Rawan Al-Nemari, Abdulrahman Al-Senaidy, Abdelhabib Semlali

Abstract:

Background and objectives: The most common cause of death in women worldwide is breast cancer. Regarding all chemotherapy disadvantages and side effects, it’s becoming necessary to identify natural products that target cancer cells with lesser harmful side effects on non-targeted cells and biological environment. Different parts of A. squamosa L., commonly known as custard apple, show varied therapeutic effects. The objective of this study is to investigate in vitro and in vivo, the anti-cancer activity of A. squamosa leaves extract. Methods: The physiological responses using MTT, nucleus staining, and LDH assays were used to evaluate cell survival and proliferation in both ER+ and ER- cells when they were exposed to extract. Monolayer wound repair assay was used to investigate the effect of extracts on cell migration. Apoptotic gene’s expression was investigated by real-time polymerase chain reaction. To study the effect of the extract on the size of tumor, breast cancer induced rats were used. Results: A. squamosa leaves extract showed high anti-proliferative and cytotoxicity effects against different breast cancer cell lines with high concentration, 100 ug/ml. The extracts have reduced the cells wound closure. Polymerase chain reaction revealed downregulation of Bcl-2 and upregulation of Bax. In breast cancer model animal developed in our laboratory, after 4 weeks treatment, treated groups have shown smaller tumor size in comparison with control group (n=4). Conclusion: These results suggest that A. squamosa leaves extract has anti-cancer activity against breast cancer in both in vitro and in vivo, and it may be developed as a potential novel agent to treat breast cancer.

Keywords: apoptosis, breast cancer, migration, proliferation

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3169 Reaction Kinetics for the Pyrolysis of Urea Phosphate

Authors: P. A. Broodryk, A. F. Van Der Merwe, H. W. J. P. Neomagus

Abstract:

The production of the clear liquid fertilizer ammonium polyphosphate (APP) is best achieved by the pyrolysis of urea phosphate, as it produces a product that is free from any of the impurities present in the raw phosphoric acid it was made from. This is a multiphase, multi-step reaction that produces carbon dioxide and ammonia as gasses and ammonium polyphosphate as liquid products. The polyphosphate chain length affects the solubility and thus the applicability of the product as liquid fertiliser, thus proper control of the reaction conditions is thus required for the use of this reaction in the production of fertilisers. This study investigates the reaction kinetics of the aforementioned reaction, describing a mathematical model for the kinetics of the reaction along with the accompanying rate constants. The reaction is initially exothermic, producing only carbon dioxide as a gas product and ammonium diphosphate, at higher temperatures the reaction becomes endothermic, producing ammonia gas as an additional by-product and longer chain polyphosphates, which when condensed too far becomes highly water insoluble. The aim of this study was to (i) characterise the pyrolysis reaction of urea phosphate by determining the mechanisms and the associated kinetic constants, and (ii) to determine the optimum conditions for ammonium diphosphate production. A qualitative investigation was also done to find the rate of hydrolysis of APP as this provides an estimate of the shelf life of an APP clear liquid fertiliser solution.

Keywords: ammonium polyphosphate, kinetics, pyrolysis, urea phosphate

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3168 Evaluation of Four Different DNA Targets in Polymerase Chain Reaction for Detection and Genotyping of Helicobacter pylori

Authors: Abu Salim Mustafa

Abstract:

Polymerase chain reaction (PCR) assays targeting genomic DNA segments have been established for the detection of Helicobacter pylori in clinical specimens. However, the data on comparative evaluations of various targets in detection of H. pylori are limited. Furthermore, the frequencies of vacA (s1 and s2) and cagA genotypes, which are suggested to be involved in the pathogenesis of H. pylori in other parts of the world, are not well studied in Kuwait. The aim of this study was to evaluate PCR assays for the detection and genotyping of H. pylori by targeting the amplification of DNA targets from four genomic segments. The genomic DNA were isolated from 72 clinical isolates of H. pylori and tested in PCR with four pairs of oligonucleotides primers, i.e. ECH-U/ECH-L, ET-5U/ET-5L, CagAF/CagAR and Vac1F/Vac1XR, which were expected to amplify targets of various sizes (471 bp, 230 bp, 183 bp and 176/203 bp, respectively) from the genomic DNA of H. pylori. The PCR-amplified DNA were analyzed by agarose gel electrophoresis. PCR products of expected size were obtained with all primer pairs by using genomic DNA isolated from H. pylori. DNA dilution experiments showed that the most sensitive PCR target was 471 bp DNA amplified by the primers ECH-U/ECH-L, followed by the targets of Vac1F/Vac1XR (176 bp/203 DNA), CagAF/CagAR (183 bp DNA) and ET-5U/ET-5L (230 bp DNA). However, when tested with undiluted genomic DNA isolated from single colonies of all isolates, the Vac1F/Vac1XR target provided the maximum positive results (71/72 (99% positives)), followed by ECH-U/ECH-L (69/72 (93% positives)), ET-5U/ET-5L (51/72 (71% positives)) and CagAF/CagAR (26/72 (46% positives)). The results of genotyping experiments showed that vacA s1 (46% positive) and vacA s2 (54% positive) genotypes were almost equally associated with VaCA+/CagA- isolates (P > 0.05), but with VacA+/CagA+ isolates, S1 genotype (92% positive) was more frequently detected than S2 genotype (8% positive) (P< 0.0001). In conclusion, among the primer pairs tested, Vac1F/Vac1XR provided the best results for detection of H. pylori. The genotyping experiments showed that vacA s1 and vacA s2 genotypes were almost equally associated with vaCA+/cagA- isolates, but vacA s1 genotype had a significantly increased association with vacA+/cagA+ isolates.

Keywords: H. pylori, PCR, detection, genotyping

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3167 Size Optimization of Microfluidic Polymerase Chain Reaction Devices Using COMSOL

Authors: Foteini Zagklavara, Peter Jimack, Nikil Kapur, Ozz Querin, Harvey Thompson

Abstract:

The invention and development of the Polymerase Chain Reaction (PCR) technology have revolutionised molecular biology and molecular diagnostics. There is an urgent need to optimise their performance of those devices while reducing the total construction and operation costs. The present study proposes a CFD-enabled optimisation methodology for continuous flow (CF) PCR devices with serpentine-channel structure, which enables the trade-offs between competing objectives of DNA amplification efficiency and pressure drop to be explored. This is achieved by using a surrogate-enabled optimisation approach accounting for the geometrical features of a CF μPCR device by performing a series of simulations at a relatively small number of Design of Experiments (DoE) points, with the use of COMSOL Multiphysics 5.4. The values of the objectives are extracted from the CFD solutions, and response surfaces created using the polyharmonic splines and neural networks. After creating the respective response surfaces, genetic algorithm, and a multi-level coordinate search optimisation function are used to locate the optimum design parameters. Both optimisation methods produced similar results for both the neural network and the polyharmonic spline response surfaces. The results indicate that there is the possibility of improving the DNA efficiency by ∼2% in one PCR cycle when doubling the width of the microchannel to 400 μm while maintaining the height at the value of the original design (50μm). Moreover, the increase in the width of the serpentine microchannel is combined with a decrease in its total length in order to obtain the same residence times in all the simulations, resulting in a smaller total substrate volume (32.94% decrease). A multi-objective optimisation is also performed with the use of a Pareto Front plot. Such knowledge will enable designers to maximise the amount of DNA amplified or to minimise the time taken throughout thermal cycling in such devices.

Keywords: PCR, optimisation, microfluidics, COMSOL

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3166 Occurrence of Porcine circovirus Type 2 in Pigs of Eastern Cape Province South Africa

Authors: Kayode O. Afolabi, Benson C. Iweriebor, Anthony I. Okoh, Larry C. Obi

Abstract:

Porcine circovirus type 2 (PCV2) is the major etiological viral agent of porcine multisystemic wasting syndrome (PWMS) and other porcine circovirus-associated diseases (PCVAD) of great economic importance in pig industry globally. In an effort to determine the status of swine herds in the Province as regarding the ‘small but powerful’ viral pathogen; a total of 375 blood, faecal and nasal swab samples were obtained from seven pig farms (commercial and communal) in Amathole, O.R. Tambo and Chris-Hani District Municipalities of Eastern Cape Province between the year 2015 and 2016. Three hundred and thirty nine (339) samples out of the total sample were subjected to molecular screening using PCV2 specific primers by conventional polymerase chain reaction (PCR). Selected sequences were further analyzed and confirmed through genome sequencing and phylogenetic analyses. The data obtained revealed that 15.93% of the screened samples (54/339) from the swine herds of the studied areas were positive for PCV2; while the severity of occurrence of the viral pathogen as observed at farm level ranges from approximately 5.6% to 60% in the studied farms. The Majority, precisely 15 out of 17 (88%) analyzed sequences were found clustering with other PCV2b reference strains in the phylogenetic analysis. More interestingly, two other sequences obtained were also found clustering within PCV2d genogroup, which is presently another fast-spreading genotype with observable higher virulence in global swine herds. This finding confirmed the presence of this all-important viral pathogen in pigs of the region; which could result in a serious outbreak of PCVAD and huge economic loss at the instances of triggering factors if no appropriate measures are taken to curb its spread effectively.

Keywords: pigs, polymerase chain reaction, porcine circovirus type 2, South Africa

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3165 Tenofovir-Amino Acid Conjugates Act as Polymerase Substrates: Implications for Avoiding Cellular Phosphorylation in the Discovery of Nucleotide Analogs

Authors: Weijie Gu, Sergio Martinez, Hoai Nguyen, Hongtao Xu, Piet Herdewijn, Steven De Jonghe, Kalyan Das

Abstract:

Nucleotide analogs are used for treating viral infections such as HIV, hepatitis B, hepatitis C, influenza, and SARS-CoV-2. To become polymerase substrates, a nucleotide analog must be phosphorylated by cellular kinases, which are rate-limiting. The goal of this study is to develop dNTP/NTP analogs directly from nucleotides. Tenofovir (TFV) analogs were synthesized by conjugating with natural or unnatural amino acids. It demonstrates that some conjugates act as dNTP analogs, and HIV-1 reverse transcriptase (RT) catalytically incorporates the TFV part as the chain terminator. X-ray structures in complex with HIV-1 RT/dsDNA showed binding of the conjugates at the polymerase active site, however, in different modes in the presence of Mg²⁺ vs. Mn²⁺ ions. The adaptability of the compounds is seemingly essential for catalytic incorporation of TFV by RT. 4d with a carboxyl sidechain demonstrated the highest incorporation. 4e showed weak incorporation and rather behaved as a dNTP-competitive inhibitor. This result advocates the feasibility of designing NTP/dNTP analogs by chemical substitutions to nucleotide analogs.

Keywords: dNTP analogs, nucleotide analogs, polymerase, tenofovir, X-ray structure

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3164 The Pathology of Bovine Rotavirus Infection in Calves That Confirmed by Enzyme Linked Immunosorbant Assay, Reverse Transcription Polymerase Chain Reaction and Real-Time RT-PCR

Authors: Shama Ranjan Barua, Tofazzal M. Rakib, Mohammad Alamgir Hossain, Tania Ferdushy, Sharmin Chowdhury

Abstract:

Rotavirus is one of the main etiologies of neonatal diarrhea in bovine calves that causes significant economic loss in Bangladesh. The present study was carried out to investigate the pathology of neonatal enteritis in calves due to bovine rotavirus infection in south-eastern part of Bangladesh. Rotavirus was identified by using ELISA, RT-PCR (Reverse Transcription Polymerase Chain Reaction), real-time RT-PCR. We examined 12 dead calves with history of diarrhea during necropsy. Among 12 dead calves, in gross examination, 6 were found with pathological changes in intestine, 5 calves had congestion of small intestine and rest one had no distinct pathological changes. Intestinal contents and/or faecal samples of all dead calves were collected and examined to confirm the presence of bovine rotavirus A using Enzyme linked immunosorbant assay (ELISA), RT-PCR and real-time RT-PCR. Out 12 samples, 5 (42%) samples revealed presence of bovine rotavirus A in three diagnostic tests. The histopathological changes were found almost exclusively limited in the small intestine. The lesions of rotaviral enteritis ranged from slight to moderate shortening (atrophy) of villi in the jejunum and ileum with necrotic crypts. The villi were blunt and covered by immature epithelial cells. Infected cells, stained with Haematoxylin and Eosin staining method, showed characteristic syncytia and eosinophilc intracytoplasmic inclusion body. The presence of intracytoplasmic inclusion bodies in enterocytes is the indication of viral etiology. The presence of rotavirus in the affected tissues and/or lesions was confirmed by three different immunological and molecular tests. The findings of histopathological changes will be helpful in future diagnosis of rotaviral infection in dead calves.

Keywords: calves, diarrhea, pathology, rotavirus

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3163 Characterization of Transcription Factors Involved in Early Defense Response during Interaction of Oil Palm Elaeis guineensis Jacq. with Ganoderma boninense

Authors: Sakeh N. Mohd, Bahari M. N. Abdul, Abdullah S. N. Akmar

Abstract:

Oil palm production generates high export earnings to many countries especially in Southeast Asian region. Infection by necrotrophic fungus, Ganoderma boninense on oil palm results in basal stem rot which compromises oil palm production leading to significant economic loss. There are no reliable disease treatments nor promising resistant oil palm variety has been cultivated to eradicate the disease up to date. Thus, understanding molecular mechanisms underlying early interactions of oil palm with Ganoderma boninense may be vital to promote preventive or control measure of the disease. In the present study, four months old oil palm seedlings were infected via artificial inoculation of Ganoderma boninense on rubber wood blocks. Roots of six biological replicates of treated and untreated oil palm seedlings were harvested at 0, 3, 7 and 11 days post inoculation. Next-generation sequencing was performed to generate high-throughput RNA-Seq data and identify differentially expressed genes (DEGs) during early oil palm-Ganoderma boninense interaction. Based on de novo transcriptome assembly, a total of 427,122,605 paired-end clean reads were assembled into 30,654 unigenes. DEGs analysis revealed upregulation of 173 transcription factors on Ganoderma boninense-treated oil palm seedlings. Sixty-one transcription factors were categorized as DEGs according to stringent cut-off values of genes with log2 ratio [Number of treated oil palm seedlings/ Number of untreated oil palm seedlings] ≥ |1.0| (corresponding to 2-fold or more upregulation) and P-value ≤ 0.01. Transcription factors in response to biotic stress will be screened out from abiotic stress using reverse transcriptase polymerase chain reaction. Transcription factors unique to biotic stress will be verified using real-time polymerase chain reaction. The findings will help researchers to pinpoint defense response mechanism specific against Ganoderma boninense.

Keywords: Ganoderma boninense, necrotrophic, next-generation sequencing, transcription factors

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3162 Identification of Mx Gene Polymorphism in Indragiri Hulu duck by PCR-RFLP

Authors: Restu Misrianti

Abstract:

The amino acid variation of Asn (allele A) at position 631 in Mx gene was specific to positive antiviral to avian viral desease. This research was aimed at identifying polymorphism of Mx gene in duck using molecular technique. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique was used to select the genotype of AA, AG and GG. There were thirteen duck from Indragiri Hulu regency (Riau Province) used in this experiment. DNA amplification results showed that the Mx gene in duck is found in a 73 bp fragment. Mx gene in duck did not show any polymorphism. The frequency of the resistant allele (AA) was 0%, while the frequency of the susceptible allele (GG) was 100%.

Keywords: duck, Mx gene, PCR, RFLP

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3161 Engineering C₃ Plants with SbtA, a Cyanobacterial Transporter, for Enhancing CO₂ Fixation

Authors: Vandana Deopanée Tomar, Gurpreet Kaur Sidhu, Panchsheela Nogia, Rajesh Mehrotra, Sandhya Mehrotra

Abstract:

The cyanobacterial CO₂ concentrating mechanism (CCM) operates to raise the levels of CO₂ in the vicinity of the main carboxylation enzyme Rubisco which is encapsulated in protein micro compartments called carboxysomes. Thus, due to the presence of CCM, cyanobacterial cells are able to work with high photosynthetic efficiency even at low Ci conditions and can accumulate 1000 folds high internal concentrations of Ci than external environment. Engineering of some useful CCM components into higher plants is one of the plausible approaches to improve their photosynthetic performance. The first step and the simplest approach for attaining this objective would be the transfer of cyanobacterial bicarbonate transporter such as SbtA to inner chloroplast envelope of C₃ plants. For this, SbtA transporter gene from Synechococcus elongatus PCC 7942 was fused to a transit peptide element to generate chimeric constructs in order to direct it to chloroplast inner envelope. Two transit peptides namely, TnaXTP (transit peptide from AT3G56160) and TMDTP (transit peptide from AT2G02590) were shortlisted from Arabidopsis thaliana genome and cloned in plant expression vector pCAMBIA1302 having mgfp5 as a reporter gene. Plant transformation was done by agro infiltration and Agrobacterium mediated co-culture. DNA, RNA, and protein were isolated from the leaves four days post infiltration, and the presence of transgene was confirmed by gene specific PCR (Polymerase Chain Reaction) analysis and by RT-PCR (Reverse Transcription Polymerase Chain Reaction). The expression was confirmed at the protein level by western blotting using anti-GFP primary antibody and horseradish peroxidase (HRP) conjugated secondary antibody. The localization of the protein was detected by confocal microscopy of isolated protoplasts. We observed chloroplastic expression for both the fusion constructs which suggest that the transit peptide sequences are capable of taking the cargo protein to the chloroplasts. These constructs are now being used to generate stable transgenic plants by Agrobacterium mediated transformation. The stability of transgene expression will be analyzed from T₀ to T₂ generation.

Keywords: agro infiltration, bicarbonate transporter, carbon concentrating mechanisms, cyanobacteria, SbtA

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3160 Detection of Polymorphism of Growth Hormone Gene in Holstein Cattle

Authors: Emine Şahin, Murat Soner Balcıoğlu

Abstract:

The aim of this study was to determine the growth hormone (bGH) gene polymorphism in the Holstein cattle growing around Antalya in Turkey. In order to determine the bGH-AluI polymorphism, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method was performed. A 891 bp fragment of bGH was amplified and two types of alleles C and D for bGH were observed. In this study, the frequencies of C and D alleles were 0.8438 and 0.1562, respectively. The genotype frequencies for CC, CD and DD were 0.787, 0.191 and 0.022, respectively. According to the results of the chi-square test, a significant deviation from the Hardy-Weinberg equilibrium was not determined for the bGH locus in the population.

Keywords: Growth Hormone Gene, Holstein , Polymorphism, RFLP

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3159 Genotyping of Rotaviruses in Pediatric Patients with Gastroenteritis by Using Real-Time Reverse Transcription Polymerase Chain Reaction

Authors: Recep Kesli, Cengiz Demir, Riza Durmaz, Zekiye Bakkaloglu, Aysegul Bukulmez

Abstract:

Objective: Acute diarrhea disease in children is a major cause of morbidity worldwide and is a leading cause of mortality, and it is the most common agent responsible for acute gastroenteritis in developing countries. With hospitalized children suffering from acute enteric disease up to 50% of the analyzed specimen were positive for rotavirus. Further molecular surveillance could provide a sound basis for improving the response to epidemic gastroenteritis and could provide data needed for the introduction of vaccination programmes in the country. The aim of this study was to investigate the prevalence of viral etiology of the gastroenteritis in children aged 0-6 years with acute gastroenteritis and to determine predominant genotypes of rotaviruses in the province of Afyonkarahisar, Turkey. Methods: An epidemiological study on rotavirus was carried out during 2016. Fecal samples obtained from the 144 rotavirus positive children with 0-6 years of ages and applied to the Pediatric Diseases Outpatient of ANS Research and Practice Hospital, Afyon Kocatepe University with the complaint of diarrhea. Bacterial agents causing gastroenteritis were excluded by using bacteriological culture methods and finally, no growth observed. Rotavirus antigen was examined by both the immunochromatographic (One Step Rotavirus and Adenovirus Combo Test, China) and ELISA (Premier Rotaclone, USA) methods in stool samples. Rotavirus RNA was detected by using one step real-time reverse transcription-polymerase chain reaction (RT-PCR). G and P genotypes were determined using RT-PCR with consensus primers of VP7 and VP4 genes, followed by semi nested type-specific multiplex PCR. Results: Of the total 144 rotavirus antigen-positive samples with RT-PCR, 4 (2,8%) were rejected, 95 (66%) were examined, and 45 (31,2%) have not been examined for PCR yet. Ninety-one (95,8%) of the 95 examined samples were found to be rotavirus positive with RT-PCR. Rotavirus subgenotyping distributions in G, P and G/P genotype groups were determined as; G1:45%, G2:27%, G3:13%, G9:13%, G4:1% and G12:1% for G genotype, and P[4]:33%, P[8]:66%, P[10]:1% for P genotype, and G1P[8]:%37, G2P[4]:%21, G3P[8]:%10, G4P[8]:%1, G9P[8]:%8, G2P[8]:%3 for G/P genotype . Not common genotype combination were %20 in G/P genotype. Conclusions: This study subscribes to the global agreement of the molecular epidemiology of rotavirus which will be useful in guiding the alternative and application of rotavirus vaccines or effective control and interception. Determining the diversity and rates of rotavirus genotypes will definitely provide guidelines for developing the most suitable vaccine.

Keywords: gastroenteritis, genotyping, rotavirus, RT-PCR

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3158 3D Printing: Rebounding from Global Supply Chain Disruption Due to Natural Disaster

Authors: Gurjinder Singh, Jasmeen Kaur, Mukul Dhiman

Abstract:

This paper mainly describes the significance of 3D printing in the supply chain management in a scenario when there is disruption in global supply chain. Furthermore, the development and implementation of supply chain strategies in context of 3D printing technology is framed to make supply chain of an organization resilient to disruption caused by natural disasters.

Keywords: 3D printing, global supply chain, supply chain management, supply chain strategies

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3157 Supply Chain Competitiveness with the Perspective of Service Performance Between Supply Chain Actors and Functions: A Theoretical Model

Authors: Umer Mukhtar

Abstract:

Supply Chain Competitiveness is the capability of a supply chain to deliver value to the customer for the sake of competitive advantage. Service Performance and Quality intervene between supply chain actors including functions inside the firm in a significant way for the supply chain to achieve a competitive position in the market to gain competitive advantage. Supply Chain competitiveness is the current issue of interest because of supply chains’ competition for competitive advantage rather than firms’. A proposed theoretical model is developed by extracting and integrating different theories to pursue further inquiry based on case studies and survey design. It is also intended to develop a scale of service performance for functions of the focal firm that is a revolving center for a whole supply chain.

Keywords: supply chain competitiveness, service performance in supply chain, service quality in supply chain, competitive advantage by supply chain, networks and supply chain, customer value, value supply chain, value chain

Procedia PDF Downloads 479