Search results for: polymerase%20chain%20reaction.

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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Authors: Virupakshaiah DBM, Madiha Ahmed, Smita T. Patil, Chandrakanth Kelmani

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Tuberculosis (TB) is a bacterial infectious disease caused by the obligate human pathogen, Mycobacterium tuberculosis. Multidrug-resistant tuberculosis (MDR-TB) is a global reality that threatens tuberculosis control. Resistance to antibiotic Rifampicin, occurs in 95% of cases through nucleotide substitutions in an 81-bp core region of the rpoB i.e; beta subunit of DNA dependant RNA polymerase. In this paper, we studied the Rifampicin-rpoB receptor interactions In silico. First, homology modeling was performed to obtain the three dimensional structure of Mycobacterium rpoB. Sixty analogs of Rifampicin were prepared using Marvin sketch software. Both original Rifampicin and the analogs were docked with rpoB and energy values were obtained. Out of sixty analogs, 43 analogs had lesser energy values than conventional Rifampicin and hence are predicted to have greater binding affinity to rpoB. Thus, this study offers a route for the development of Rifampicin analogs against multi drug resistant Mycobacterium rpoB.

Keywords: Marvin Sketch, Mycobacterium tuberculosis, Rifampicin, rpoB.

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Authors: M. Seifi, S. Fallah, M. Firoozrai

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Matrix metalloproteinase-3 (MMP3) is key member of the MMP family, and is known to be present in coronary atherosclerotic. Several studies have demonstrated that MMP-3 5A/6A polymorphism modify each transcriptional activity in allele specific manner. We hypothesized that this polymorphism may play a role as risk factor for development of coronary stenosis. The aim of our study was to estimate MMP-3 (5A/6A) gene polymorphism on interindividual variability in risk for coronary stenosis in an Iranian population.DNA was extracted from white blood cells and genotypes were obtained from coronary stenosis cases (n=95) and controls (n=100) by PCR (polymerase chain reaction) and restriction fragment length polymorphism techniques. Significant differences between cases and controls were observed for MMP3 genotype frequencies (X2=199.305, p< 0.001); the 6A allele was less frequently seen in the control group, compared to the disease group (85.79 vs. 78%, 6A/6A+5A/6A vs. 5A/5A, P≤0.001). These data imply the involvement of -1612 5A/6A polymorphism in coronary stenosis, and suggest that probably the 6A/6A MMP-3 genotype is a genetic susceptibility factor for coronary stenosis.

Keywords: Coronary artery stenosis, matrixmetalloproteinase-3, polymorphism, polymerase chain reaction.

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Authors: Sin J. Chen, Jyh J. Chen

Abstract:

This study describes a micro device integrated with multi-chamber for polymerase chain reaction (PCR) with different annealing temperatures. The device consists of the reaction polydimethylsiloxane (PDMS) chip, a cover glass chip, and is equipped with cartridge heaters, fans, and thermocouples for temperature control. In this prototype, commercial software is utilized to determine the geometric and operational parameters those are responsible for creating the denaturation, annealing, and extension temperatures within the chip. Two cartridge heaters are placed at two sides of the chip and maintained at two different temperatures to achieve a thermal gradient on the chip during the annealing step. The temperatures on the chip surface are measured via an infrared imager. Some thermocouples inserted into the reaction chambers are used to obtain the transient temperature profiles of the reaction chambers during several thermal cycles. The experimental temperatures compared to the simulated results show a similar trend. 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, temperature gradient, micro-fabrication.

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Authors: Cheng-Hong Yang, Yu-Huei Cheng, Hsueh-Wei Chang, Li-Yeh Chuang

Abstract:

Before performing polymerase chain reactions (PCR), a feasible primer set is required. Many primer design methods have been proposed for design a feasible primer set. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this paper, a particle swarm optimization (PSO) algorithm is proposed to solve primer design problems associated with providing a specific product for PCR experiments. A test set of the gene CYP1A1, associated with a heightened lung cancer risk was analyzed and the comparison of accuracy and running time with the genetic algorithm (GA) and memetic algorithm (MA) was performed. A comparison of results indicated that the proposed PSO method for primer design finds optimal or near-optimal primer sets and effective PCR products in a relatively short time.

Keywords: polymerase chain reaction (PCR), primer design, evolutionary computation, particle swarm optimization (PSO).

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

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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: Zika Virus, polymerase chain reaction, microcephaly, amniotic fluid.

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Authors: Tse-Yu Hsieh, Jyh-Jian Chen

Abstract:

Since polymerase chain reaction (PCR) has been invented, it has emerged as a powerful tool in genetic analysis. The PCR products are closely linked with thermal cycles. Therefore, to reduce the reaction time and make temperature distribution uniform in the reaction chamber, a novel oscillatory thermal cycler is designed. The sample is placed in a fixed chamber, and three constant isothermal zones are established and lined in the system. The sample is oscillated and contacted with three different isothermal zones to complete thermal cycles. This study presents the design of the geometric characteristics of the chamber. The commercial software CFD-ACE+TM is utilized to investigate the influences of various materials, heating times, chamber volumes, and moving speed of the chamber on the temperature distributions inside the chamber. The chamber moves at a specific velocity and the boundary conditions with time variations are related to the moving speed. Whereas the chamber moves, the boundary is specified at the conditions of the convection or the uniform temperature. The user subroutines compiled by the FORTRAN language are used to make the numerical results realistically. Results show that the reaction chamber with a rectangular prism is heated on six faces; the effects of various moving speeds of the chamber on the temperature distributions are examined. Regarding to the temperature profiles and the standard deviation of the temperature at the Y-cut cross section, the non-uniform temperature inside chamber is found as the moving speed is larger than 0.01 m/s. By reducing the heating faces to four, the standard deviation of the temperature of the reaction chamber is under 1.4×10-3K with the range of velocities between 0.0001 m/s and 1 m/s. The nature convective boundary conditions are set at all boundaries while the chamber moves between two heaters, the effects of various moving velocities of the chamber on the temperature distributions are negligible at the assigned time duration.

Keywords: Polymerase chain reaction, oscillatory thermal cycler, standard deviation of temperature, nature convective.

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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, detection, genotyping, Kuwait.

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Authors: M. S. Muhammad, S. M. W. Masra, K. Kipli, N. Zamhari

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The capturing of gel electrophoresis image represents the output of a DNA computing algorithm. Before this image is being captured, DNA computing involves parallel overlap assembly (POA) and polymerase chain reaction (PCR) that is the main of this computing algorithm. However, the design of the DNA oligonucleotides to represent a problem is quite complicated and is prone to errors. In order to reduce these errors during the design stage before the actual in-vitro experiment is carried out; a simulation software capable of simulating the POA and PCR processes is developed. This simulation software capability is unlimited where problem of any size and complexity can be simulated, thus saving cost due to possible errors during the design process. Information regarding the DNA sequence during the computing process as well as the computing output can be extracted at the same time using the simulation software.

Keywords: DNA computing, PCR, POA, simulation software

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Authors: Wiriya Loongyai, Kiettisak Promphet, Nilubol Kangsukul, Ratchawat Noppha

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Polymerase chain reaction (PCR) assay and conventional microbiological methods were used to detect bacterial contamination of egg shells and egg content in different commercial housing systems, open house system and evaporative cooling system. A PCR assay was developed for direct detection using a set of primers specific for the invasion by A gene (invA) of Salmonella spp. PCR detected the presence of Salmonella in 2 samples of shell egg from the evaporative cooling system, while conventional cultural methods detected no Salmonella from the same samples.

Keywords: egg content, egg shell, invA gene, PCR, Salmonellaspp.

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Authors: Mustafa Yorgancılar, Emine Atalay, Necdet Akgün, Ali Topal

Abstract:

In this study, polymerase chain reaction based Inter-simple sequence repeat (ISSR) from DNA fingerprinting techniques were used to investigate the genetic relationships among barley crossbreed genotypes in Turkey. It is important that selection based on the genetic base in breeding programs via ISSR, in terms of breeding time. 14 ISSR primers generated a total of 97 bands, of which 81 (83.35%) were polymorphic. The highest total resolution power (RP) value was obtained from the F2 (0.53) and M16 (0.51) primers. According to the ISSR result, the genetic similarity index changed between 0.64–095; Lane 3 with Line 6 genotypes were the closest, while Line 36 were the most distant ones. The ISSR markers were found to be promising for assessing genetic diversity in barley crossbreed genotypes.

Keywords: Barley, crossbreed, genetic similarity, ISSR.

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Authors: F. S. Abtahi, M. Koohi Hbibi, M. Khodaei Motlagh

Abstract:

Bean common mosaic necrosis virus (BCMNV) is a potyvirus with a worldwide distribution. This virus causes serious economic losses in Iran in many leguminoses. During 20008, samples were collected from soybeans fields in Tehran Province. Four isolates (S1, S2 and S3) were inoculated on 15 species of Cucurbitaceae, Chenopodiaceae, Solanacae and Leguminosae. Chenopodium quinoa and C. amaranticolor. Did not developed any symptoms.all isolates caused mosaic symptoms on Phaseolus vulgaris cv. Red Kidney and P. vulgaris cv. Bountiful. The molecular weights of coat protein using SDS-PAGE and western blotting were estimated at 33 kDa. Reverse transcription polymerase chain reaction (RT-PCR) was performed using one primer pairs designed by L. XU et al. An approximately 920 bp fragment was amplified with a specific primer.

Keywords: ELISA, RT-PCR, SDS-PAGE, BCMNV.

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Authors: Öznur Özge Özcan, Canan Sercan, Hamza Kulaksız, Mesut Karahan, Korkut Ulucan

Abstract:

Genetic structure is very important to understand the brain dopamine system which is related to athletic performance. Hopefully, there will be enough studies about athletics performance in the terms of addiction-related genetic markers in the future. In the present study, we intended to investigate the Receptor-2 Gene (DRD2) rs1800497, which is related to brain dopaminergic system. 10 sprinter and 10 endurance athletes were enrolled in the study. Real-Time Polymerase Chain Reaction method was used for genotyping. According to results, A1A1, A1A2 and A2A2 genotypes in athletes were 0 (%0), 3 (%15) and 17 (%85). A1A1 genotype was not found and A2 allele was counted as the dominating allele in our cohort. These findings show that dopaminergic mechanism effects on sport genetic may be explained by the polygenic and multifactorial view.

Keywords: Addiction, athletic performance, genotype, polymorphism, sport genetics.

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Authors: S. Moghaddaszadeh-Ahrabi, S. Farajnia, Gh. Rahimi-Mianji, A. Nejati-Javaremi

Abstract:

In recent years, there has been an increasing interest toward the use of bovine genotyped embryos for commercial embryo transfer programs. Biopsy of a few cells in morulla stage is essential for preimplantation genetic diagnosis (PGD). Low amount of DNA have limited performing the several molecular analyses within PGD analyses. Whole genome amplification (WGA) promises to eliminate this problem. We evaluated the possibility and performance of an improved primer extension preamplification (I-PEP) method with a range of starting bovine genomic DNA from 1-8 cells into the WGA reaction. We optimized a short and simple I-PEP (ssI-PEP) procedure (~3h). This optimized WGA method was assessed by 6 loci specific polymerase chain reactions (PCRs), included restriction fragments length polymorphism (RFLP). Optimized WGA procedure possesses enough sensitivity for molecular genetic analyses through the few input cells. This is a new era for generating characterized bovine embryos in preimplantation stage.

Keywords: Whole genome amplification (WGA), Genotyping, Bovine, Preimplantation genetic diagnosis (PGD)

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Authors: S. Fallah, M. Seifi, M. Firoozrai, T. Godarzi, M. Jafarzadeh, L. H. Ghohari

Abstract:

The ε4 allele of the ε2, ε3 and ε4 protein isoform polymorphism in the gene encoding apolipoprotein E (Apo E) has previously been associated with increased cardiac artery disease (CAD); therefore to investigate the significance of this polymorphism in pathogenesis of CAD in Iranian patients with stenosis and control subjects. To investigate the association between  Apo E polymorphism and coronary artery disease we performed a comparative case control study of the frequency of Apo E  polymorphism in One hundred CAD patients with stenosis who underwent coronary angiography (>50% stenosis) and 100 control subjects (<10% stenosis). The Apo E alleles and genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). We observed an association between the Apo E polymorphism and CAD in this study. These data suggest that the Apo ε4 and ε2 alleles increase the risk for CAD in Iranian population (χ2 =4.26, p= 0.05, OR=2 and χ2 =0.38, p=0.53, OR=1.2). These results suggest that ε4 and ε2 alleles are risk factors for stenosis.

Keywords: Arterial blood vessels, atherosclerosis, cholesterol.

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Authors: N. Hussain, G. Jaffery, A.N. Sabri, S. Hasnain

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The main aim is to perform mutational analysis of CTLA4 gene Exon 1 in SLE patients. A total of 61 SLE patients fulfilling “American College of Rheumatology (ACR) criteria" and 61 controls were enrolled in this study. The region of CTLA4 gene exon 1 was amplified by using Step-down PCR technique. Extracted DNA of band 354 bp was sequenced to analyze mutations in the exon-1 of CTLA-4 gene. Further, protein sequences were identified from nucleotide sequences of CTLA4 Exon 1 by using Expasy software and through Blast P software it was found that CTLA4 protein sequences of Pakistani SLE patients were similar to that of Chinese SLE population. No variations were found after patients sequences were compared with that of the control sequence. Furthermore it was found that CTLA4 protein sequences of Pakistani SLE patients were similar to that of Chinese SLE population. Thus CTLA4 gene may not be responsible for an autoimmune disease SLE.

Keywords: American College of Rheumatology criteria, autoimmune disease, Cytotoxic T Lymphocyte Antigen-4, Polymerase Chain Reaction, Systemic Lupus Erythematosus

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Authors: Kritsada Khongnomnan, Wittaya Poomipak, Yong Poovorawan, Sunchai Payungporn

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In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) which naturally infected human were analyzed by bioinformatic approaches to find candidate human cellular miRNAs targeting viral genomes. There were 76 miRNAs targeting influenza A viruses. Among these candidates, 70 miRNAs were subtypes specifically targeting each subtype of influenza A virus including 21 miRNAs targeted subtype H1N1, 27 miRNAs targeted subtype H5N1 and 22 miRNAs targeted subtype H3N2. The remaining 6 miRNAs target on multiple subtypes of influenza A viruses. Uniquely, hsa-miR-3145 is the only one candidate miRNA targeting PB1 gene of all three subtypes. Obviously, most of the candidate miRNAs are targeting on polymerase complex genes (PB2, PB1 and PA) of influenza A viruses. This study predicted potential human miRNAs targeting on different subtypes of influenza A viruses which might be useful for inhibition of viral replication and for better understanding of the interaction between virus and host cell.

Keywords: Human miRNAs, Influenza A viruses, H1N1, H5N1, H3N2

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Authors: Archana Panchapakesan Iyer, Taha Abdullah Kumosani

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Many high-risk pathogens that cause disease in humans are transmitted through various food items. Food-borne disease constitutes a major public health problem. Assessment of the quality and safety of foods is important in human health. Rapid and easy detection of pathogenic organisms will facilitate precautionary measures to maintain healthy food. The Polymerase Chain Reaction (PCR) is a handy tool for rapid detection of low numbers of bacteria. We have designed gene specific primers for most common food borne pathogens such as Staphylococci, Salmonella and E.coli. Bacteria were isolated from food samples of various food outlets and identified using gene specific PCRs. We identified Staphylococci, Salmonella and E.coli O157 using gene specific primers by rapid and direct PCR technique in various food samples. This study helps us in getting a complete picture of the various pathogens that threaten to cause and spread food borne diseases and it would also enable establishment of a routine procedure and methodology for rapid identification of food borne bacteria using the rapid technique of direct PCR. This study will also enable us to judge the efficiency of present food safety steps taken by food manufacturers and exporters.

Keywords: food borne pathogens, PCR, food safety, rapiddetection.

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

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Halal authentication and verification in supplement capsules are highly required as the gelatine available in the market can be from halal or non-halal sources. It is an obligation for Muslim to consume and use the halal consumer goods. At present, real-time polymerase chain reaction (RT-PCR) is the most common technique being used for the detection of porcine and bovine DNA in gelatine due to high sensitivity of the technique and higher stability of DNA compared to protein. In this study, twenty samples of supplements capsules from different products with different Halal logos were analyzed for porcine and bovine DNA using RT-PCR. Standard bovine and porcine gelatine from eurofins at a range of concentration from 10^-1 to 10^-5 ng/µl were used to determine the linearity range, limit of detection and specificity on RT-PCR (SYBR Green method). RT-PCR detected porcine (two samples), bovine (four samples) and mixture of porcine and bovine (six samples). The samples were also tested using FT-IR technique where normalized peak of IR spectra were pre-processed using Savitsky Golay method before Principal Components Analysis (PCA) was performed on the database. Scores plot of PCA shows three clusters of samples; bovine, porcine and mixture (bovine and porcine). The RT-PCR and FT-IR with chemometrics technique were found to give same results for porcine gelatine samples which can be used for Halal authentication.

Keywords: Halal, real-time PCR, gelatin, FTIR and chemometrics.

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Authors: Miguel García-Ferrús, Ana González, María A. Ferrús

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The most demanded organic foods worldwide are those that are consumed fresh, such as fruits and vegetables. However, there is a knowledge gap about some aspects of organic food microbiological quality and safety. Organic fruits and vegetables are more exposed to pathogenic microorganisms due to surface contact with natural fertilizers such as animal manure, wastes and vermicompost used during farming. Therefore, the objective of this work was to study the contamination of organic fresh green leafy vegetables by two emergent pathogens, Arcobacter spp. and Helicobacter pylori. For this purpose, a total of 24 vegetable samples, 13 lettuce and 11 spinach were acquired from 10 different ecological supermarkets and greengroceries and analyzed by culture and PCR. Arcobacter spp. was detected in five samples (20%) by PCR, four spinach and one lettuce. One spinach sample was found to be also positive by culture. For H. pylori, the H. pylori VacA gene-specific band was detected in 12 vegetable samples (50%), 10 lettuces and two spinach. Isolation in the selective medium did not yield any positive result, possibly because of low contamination levels together with the presence of the organism in its viable but non-culturable form. Results showed significant levels of H. pylori and Arcobacter contamination in organic vegetables that are generally consumed raw, which seems to confirm that these foods can act as transmission vehicles to humans.

Keywords: Arcobacter spp., Helicobacter pylori, organic vegetables, Polymerase Chain Reaction, PCR.

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Authors: Hai Xu, Yiwei Wang, Xi Bao, Bihua Deng, Pengcheng Li, Yu Lu

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T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 10¹⁰ pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac^®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.

Keywords: Gonadotrophin releasing hormone, GnRH, immunocastration, T7 phage, phage vaccine.

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Authors: Yan Ren, Jun Qiao, Xianxia Liu, Pengyan Wang, Qiang Fu, Huijun Shi, Fei Guo, Yuanzhi Wang, Hui Zhang, Jinliang Sheng, Xinli Gu, Xiao-Jun Liu, Chuangfu Chen

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As part of national epidemiological survey on bovine viral diarrhea virus (BVDV), a total of 274 dejecta samples were collected from 14 cattle farms in 8 areas of Xinjiang Uygur Autonomous Region in northwestern China. Total RNA was extracted from each sample, and 5--untranslated region (UTR) of BVDV genome was amplified by using two-step reverse transcriptase-polymerase chain reaction (RT-PCR). The PCR products were subsequently sequenced to study the genetic variations of BVDV in these areas. Among the 274 samples, 33 samples were found virus-positive. According to sequence analysis of the PCR products, the 33 samples could be arranged into 16 groups. All the sequences, however, were highly conserved with BVDV Osloss strains. The virus possessed theses sequences belonged to BVDV-1b subtype by phylogenetic analysis. Based on these data, we established a typing tree for BVDV in these areas. Our results suggested that BVDV-1b was a predominant subgenotype in northwestern China and no correlation between the genetic and geographical distances could be observed above the farm level.

Keywords: bovine viral diarrhea virus, molecular epidemiology, phylogenetic analysis.

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Authors: Mohammad Ahmadinejad, Mohammad Reza Shakibaie, Kyvan Shams, Mohammad Khalili

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Legionella pneumophila is involved in more than 95% cases of severe atypical pneumonia. Infection is mainly by inhalation the indoor aerosols through the water-coolant systems. Because some Legionella strains may be viable but not culturable, therefore, Taq polymerase, DNA amplification and semi-nested-PCR were carried out to detect Legionella-specific 16S-rDNA sequence. For this purpose, 1.5 litter of water samples from 77 water-coolant system were collected from four different hospitals, two nursing homes and one student hostel in Kerman city of Iran, each in a brand new plastic bottle during summer season of 2006 (from April to August). The samples were filtered in the sterile condition through the Millipore Membrane Filter. DNA was extracted from membrane and used for PCR to detect Legionella spp. The PCR product was then subjected to semi-nested PCR for detection of L. pneumophila. Out of 77 water samples that were tested by PCR, 30 (39%) were positive for most species of Legionella. However, L. pneumophila was detected from 14 (18.2%) water samples by semi-nested PCR. From the above results it can be concluded that water coolant systems of different hospitals and nursing homes in Kerman city of Iran are highly contaminated with L. pneumophila spp. and pose serious concern. So, we recommend avoiding such type of coolant system in the hospitals and nursing homes.

Keywords: Legionella pneumophila, water-coolant system, semi-nested -PCR.

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Authors: Amani Ashari, Julia Omar, Arif Hashim, Shahrul Hamid

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Apolipoprotein E (APOE) gene polymorphism has influence on serum lipids which relates to cardiovascular risk. The purpose of this study was to determine the frequency distribution of APOE alleles among Malaysian Type 2 Diabetes Mellitus (DM) patients with and without coronary artery disease (CAD) and their association with serum lipid profiles. A total of 115 patients were recruited in which 78 patients had Type 2 DM without CAD and 37 patients had Type 2 DM with CAD. The APOE polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The APOE ɛ3 allele was the most common one in both groups. There was no significant association between the APOE genotypes and the CAD status in Type 2 DM using Pearson χ² test. Further analysis indicated there were no significant differences in all lipid parameters between E2, E3 and E4 subgroups in both groups. The study showed that the E4 allele carriers of Type 2 DM with CAD patients had higher LDL-C level and lower HDL-C level compared to the other allele carriers. However, analyses showed these levels were not statistically different. The study also showed that the Type 2 DM with CAD group with E2 allele had higher triglyceride (TG). In conclusion, further study with larger sample size is needed to confirm role of E4 as a marker of CAD among Type 2 DM patients in Malaysian population.

Keywords: Apolipoprotein E, diabetes mellitus, cardiovascular disease, lipids.

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Authors: Abimbola M. Enitan, John O. Odiyo, Feroz M. Swalaha

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The study of microbial ecology and their function in anaerobic digestion processes are essential to control the biological processes. This is to know the symbiotic relationship between the microorganisms that are involved in the conversion of complex organic matter in the industrial wastewater to simple molecules. In this study, diversity and quantity of bacterial community in the granular sludge taken from the different compartments of a full-scale upflow anaerobic sludge blanket (UASB) reactor treating brewery wastewater was investigated using polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR). The phylogenetic analysis showed three major eubacteria phyla that belong to Proteobacteria, Firmicutes and Chloroflexi in the full-scale UASB reactor, with different groups populating different compartment. The result of qPCR assay showed high amount of eubacteria with increase in concentration along the reactor’s compartment. This study extends our understanding on the diverse, topological distribution and shifts in concentration of microbial communities in the different compartments of a full-scale UASB reactor treating brewery wastewater. The colonization and the trophic interactions among these microbial populations in reducing and transforming complex organic matter within the UASB reactors were established.

Keywords: Bacteria, brewery wastewater, real-time quantitative PCR, UASB reactor.

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Authors: Ahmed F. Azmy , Amal E. Saafan, Tamer M. Essam, Magdy A. Amin, Shaban H. Ahmed

Abstract:

Bacterial strains capable of degradation of malathion from the domestic sewage were isolated by an enrichment culture technique. Three bacterial strains were screened and identified as Acinetobacter baumannii (AFA), Pseudomonas aeruginosa (PS1), and Pseudomonas mendocina (PS2) based on morphological, biochemical identification and 16S rRNA sequence analysis. Acinetobacter baumannii AFA was the most efficient malathion degrading bacterium, so used for further biodegradation study. AFA was able to grow in mineral salt medium (MSM) supplemented with malathion (100 mg/l) as a sole carbon source, and within 14 days, 84% of the initial dose was degraded by the isolate measured by high performance liquid chromatography. Strain AFA could also degrade other organophosphorus compounds including diazinon, chlorpyrifos and fenitrothion. The effect of different culture conditions on the degradation of malathion like inoculum density, other carbon or nitrogen sources, temperature and shaking were examined. Degradation of malathion and bacterial cell growth were accelerated when culture media were supplemented with yeast extract, glucose and citrate. The optimum conditions for malathion degradation by strain AFA were; an inoculum density of 1.5x 10^12CFU/ml at 30°C with shaking. A specific polymerase chain reaction primers were designed manually using multiple sequence alignment of the corresponding carboxylesterase enzymes of Acinetobacter species. Sequencing result of amplified PCR product and phylogenetic analysis showed low degree of homology with the other carboxylesterase enzymes of Acinetobacter strains, so we suggested that this enzyme is a novel esterase enzyme. Isolated bacterial strains may have potential role for use in bioremediation of malathion contaminated.

Keywords: Acinetobacter baumannii, biodegradation, Malathion, organophosphate pesticides.

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Authors: Hai-Ying Liu, Yi-Hao Chen, Shu-Yin Pang, Feng-Hua Wang, Xiao-Fang Peng, Li-Yuan Yang, Zheng-Rong Chen, Yi Chen, Bing Zhu

Abstract:

Objective: Biliary atresia (BA) is characterized by progressive liver fibrosis. Epithelial-mesenchymal transition (EMT) has been implicated as a key mechanism in the pathogenesis of organ fibrosis. MiR-200a has been shown to repress EMT. We aim to explore the role of miR-200a in the fibrogenesis of BA. Methods: We obtained the plasma samples and liver samples from patients with BA or controls to examine the role of miR-200a. Histological liver fibrosis was assessed using the Ishak fibrosis scores. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of miR-200a in plasma. We also evaluated the expression of miR-200a in liver tissues using tyramide signal amplification fluorescence in situ hybridization (TSA-FISH). The expression of EMT related proteins zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin and α-smooth muscle actin (α-SMA) in the liver sections were detected by immunohistochemical staining. Results: We found that the expression of miR-200a was both elevated in the plasma and liver tissues from BA patients compared with the controls. The hepatic expression of ZEB1 and α-SMA were markedly increased in the liver sections from BA patients compared to the controls, whereas E-cadherin was downregulated in the BA group. Simultaneously, we noted that the hepatic expression of miR-200a, E-cadherin and α-SMA were upregulated with the progression of liver fibrosis in the BA group, while ZEB1 was downregulated with the progression of liver fibrosis in BA patients. Conclusion: These findings suggest EMT has a critical effect on the fibrotic process of BA, and the interaction between miR-200a and ZEB1 may regulate EMT and eventually influence liver fibrogenesis of BA.

Keywords: Biliary atresia, liver fibrosis, MicroRNA, epithelial-mesenchymal transition, zinc finger e-box-binding homeobox 1.

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Authors: M. Marjaneh, M. Y. Narazah, H. Shahrul

Abstract:

Array-based gene expression analysis is a powerful tool to profile expression of genes and to generate information on therapeutic effects of new anti-cancer compounds. Anti-apoptotic effect of thymoquinone was studied in MCF7 breast cancer cell line using gene expression profiling with cDNA microarray. The purity and yield of RNA samples were determined using RNeasyPlus Mini kit. The Agilent RNA 6000 NanoLabChip kit evaluated the quantity of the RNA samples. AffinityScript RT oligo-dT promoter primer was used to generate cDNA strands. T7 RNA polymerase was used to convert cDNA to cRNA. The cRNA samples and human universal reference RNA were labelled with Cy-3-CTP and Cy-5-CTP, respectively. Feature Extraction and GeneSpring softwares analysed the data. The single experiment analysis revealed involvement of 64 pathways with up-regulated genes and 78 pathways with downregulated genes. The MAPK and p38-MAPK pathways were inhibited due to the up-regulation of PTPRR gene. The inhibition of p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of p38-MAPK caused up-regulation of TP53 and down-regulation of Bcl2 genes indicating involvement of intrinsic apoptotic pathway. Down-regulation of CARD16 gene as an adaptor molecule regulated CASP1 and suggested necrosis-like programmed cell death and involvement of caspase in apoptosis. Furthermore, down-regulation of GPCR, EGF-EGFR signalling pathways suggested reduction of ER. Involvement of AhR pathway which control cytochrome P450 and glucuronidation pathways showed metabolism of Thymoquinone. The findings showed differential expression of several genes in apoptosis pathways with thymoquinone treatment in estrogen receptor-positive breast cancer cells.

Keywords: CARD16, CASP10, cDNA microarray, PTPRR, Thymoquinone.

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Authors: C. Kridtayopas, W. Danvilai, P. Sopannarath, A. Kayan, W. Loongyai

Abstract:

Both Betong chicken (KU Line) and Thai Native chickens were the high quality of the meat and low carcass fat compared to broiler chickens. The objective of this study was to determine the growth performance, carcass characteristic, meat quality and association of polymorphism in the ApoVLDL-II gene with fat accumulation in the female broiler, Thai Native and Betong (KU line) chickens at 4-14 weeks. The chickens were used and reared under the same environment and management (100 chicks per breed). The results showed that body weight (BW) of broiler chickens was significantly higher than Thai Native and Betong (KU line) chickens (P < 0.01) through all the experiment. At 4-8 weeks of age, feed conversion ratio (FCR) of broiler chickens was significantly better than Thai Native and Betong (KU line) chickens (P < 0.01), then increased at week 8-14. The percentage of breast, abdominal fat and subcutaneous fat of broiler chickens was significantly greater than Thai Native and Betong (KU line) chickens (P < 0.01). However, Thai Native chickens showed the highest percentage of liver (P < 0.01) when compared to other breeds. In addition, the percentage of wing of Thai Native and Betong (KU line) chickens were significantly (P < 0.01) higher than broiler chickens. Meat quality was also determined and found that, pH of breast meat left from slaughter 45 minutes (pH45) and 24 hours (pH24) of broiler was significantly higher than Thai Native and Betong (KU line) (P < 0.01) whereas the percentage of drip loss, thawing loss, cooking loss and shear force was not significantly different between breeds. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to genotype the polymorphism in the ApoVLDL-II gene in the broiler, Thai Native and Betong (KU line) chickens. The results found that, the polymorphism in the ApoVLDL-II gene at VLDL6 loci was not associated with fat accumulation in those studied population.

Keywords: ApoVLDL-II Gene, Betong (KU line) chickens, broiler chickens, carcass characteristic, growth performance, meat quality, Thai Native Chickens.

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Authors: Sadaf Moeez, Madiha Khalid, Zoya Khalid, Sania Shaheen, Sumbul Khalid

Abstract:

Background: Inter-individual variation in response to metformin, which has been considered as a first line therapy for T2DM treatment is considerable. In the current study, it was aimed to investigate the impact of two genetic variants Leu125Phe (rs77474263) and Gly64Asp (rs77630697) in gene SLC47A1 on the clinical efficacy of metformin in T2DM Pakistani patients. Methods: The study included 800 T2DM patients (400 metformin responders and 400 metformin non-responders) along with 400 ethnically matched healthy individuals. The genotypes were determined by allele-specific polymerase chain reaction. In-silico analysis was done to confirm the effect of the two SNPs on the structure of genes. Association was statistically determined using SPSS software. Results: Minor allele frequency for rs77474263 and rs77630697 was 0.13 and 0.12. For SLC47A1 rs77474263 the homozygotes of one mutant allele ‘T’ (CT) of rs77474263 variant were fewer in metformin responders than metformin non-responders (29.2% vs. 35.5 %). Likewise, the efficacy was further reduced (7.2% vs. 4.0 %) in homozygotes of two copies of ‘T’ allele (TT). Remarkably, T2DM cases with two copies of allele ‘C’ (CC) had 2.11 times more probability to respond towards metformin monotherapy. For SLC47A1 rs77630697 the homozygotes of one mutant allele ‘A’ (GA) of rs77630697 variant were fewer in metformin responders than metformin non-responders (33.5% vs. 43.0 %). Likewise, the efficacy was further reduced (8.5% vs. 4.5%) in homozygotes of two copies of ‘A’ allele (AA). Remarkably, T2DM cases with two copies of allele ‘G’ (GG) had 2.41 times more probability to respond towards metformin monotherapy. In-silico analysis revealed that these two variants affect the structure and stability of their corresponding proteins. Conclusion: The present data suggest that SLC47A1 Leu125Phe (rs77474263) and Gly64Asp (rs77630697) polymorphisms were associated with the therapeutic response of metformin in T2DM patients of Pakistan.

Keywords: Diabetes, T2DM, SLC47A1, Pakistan, polymorphism.

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