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.
Abstract: 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.
Abstract: Meat Tenderness is one of the most important factors affecting consumers' assessment of meat quality. Variation in meat tenderness is genetically controlled and varies among breeds, and it is also influenced by environmental factors that can affect its creation during rigor mortis and postmortem. The final postmortem meat tenderization relies on the extent of proteolysis of myofibrillar proteins caused by the endogenous activity of the proteolytic calpain system. This calpain system includes different calcium-dependent cysteine proteases, and an inhibitor, calpastatin. It is widely accepted that in farm animals including chickens, the μ-calpain gene (CAPN1) is a physiological candidate gene for meat tenderness. This study aimed to identify the association of single nucleotide polymorphism (SNP) markers in the CAPN1 gene with the tenderness of chicken breast meat from two Malaysian native and commercial broiler breed crosses. Ten, five months old native chickens and ten, 42 days commercial broilers were collected from the local market and breast muscles were removed two hours after slaughter, packed separately in plastic bags and kept at -20ºC for 24 h. The tenderness phenotype for all chickens’ breast meats was determined by Warner-Bratzler Shear Force (WBSF). Thawing and cooking losses were also measured in the same breast samples before using in WBSF determination. Polymerase chain reaction (PCR) was used to identify the previously reported C7198A and G9950A SNPs in the CAPN1 gene and assess their associations with meat tenderness in the two breeds. The broiler breast meat showed lower shear force values and lower thawing loss rates than the native chickens (p
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 (
Abstract: 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.
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.
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.
Abstract: 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.
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.