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This paper reports an outdoor-use polymerase chain reaction (PCR) technology in which stability of resistance temperature detectors (RTDs) is remarkably improved. A thin-film RTD made of non-annealed Pt shows accuracy degradation because the resistance of the RTD tends to decrease during the PCR operation. Thus, the annealing process is applied to the Pt RTD to improve the stability, which is a prerequisite to the accurate measurement of the absolute temperature. Both heaters and the RTD are fabricated on a thin quartz substrate whose melting temperature is high enough for annealing. The performances in the PCR time and power consumption are enhanced by reducing the size of the heater chips with no degradation in the temperature uniformity. A spring-loaded electrode is employed to simplify the procedure of electrical connection to the thermal controller and loading/unloading of the PCR chip. The contact area of the electrical connection is so small that the conductive thermal resistance increases; thereby small heat dissipation can be exploited for low-power operation. The stability of the RTD is experimentally confirmed in terms of resistance variation over repeated PCR operations (four times). The least variation of 0.005%, which corresponds to a negligible temperature variation of 0.038 °C for the PCR, is achieved from the RTD annealed for 5 min at 450 °C. The gel-electrophoresis result indicates that the PCR performance of the proposed system using a film-type PCR chip is comparable to that of a conventional system using a vial tube despite its low power consumption., Competing Interests: The authors have declared that no competing interests exist.

The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Scopulariopsis brevicaulis is known as the most common etiological factor of the mould toenail infections. There are also reports indicating that S. brevicaulis could cause organ and disseminated infections. Nowadays microscopic observations from the direct sample and culture are crucial for the appropriate recognition of the infection. In this paper a PCR-based method for S. brevicaulis detection is presented. The specificity of the reaction was confirmed, as positive results were obtained only for tested S. brevicaulis isolates and no positive results were obtained for other moulds, dermatophytes, yeast-like fungi, and human DNA.

The genus Saccharomyces comprises seven single-genome species (S. arboricola, S. cerevisiae, S. eubayanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum) and two hybrid species - S. pastorianus (S. cerevisiae plus S. eubayanus) and S. bayanus (mostly S. uvarum plus S. eubayanus). Species-specific primers have already been developed for the identification of each of the single-genome species, and these primers can usually detect both genomes in hybrids. It would be advantageous if a single reaction could detect any member of the clade. We have investigated three potentially generic approaches to design genus-specific primers. Two methods that both use sequence alignment differences for primer design were only partly successful. A third method used synteny data to identify 136 target genes that are potentially present only in all species of the Saccharomyces clade. HSP30 (YCR021C) was fully successful; different primer pairs were developed with high G+C content for use at 63 °C. In < 3 h, using a robust colony-PCR followed by gel electrophoresis, the method can reliably detect any member of the genus. This novel approach still uses conventional sequence alignment mismatches but relies principally on the presence of the target gene only within the genus Saccharomyces., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Objective: To determine the frequency of mixed donor chimerism in patients of non-malignant haematological diseases after allogeneic bone marrow transplant., Study Design: A cross-sectional, observational study., Place and Duration of Study: Department of Haematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from July 2010 to June 2011., Methodology: Donor chimerism was assessed in patients of aplastic anaemia and beta-thalassaemia major who underwent allogeneic bone marrow transplantation (BMT). Peripheral blood samples were used to assess chimerism status by analysis of short tandem repeats (STR). In patients where pre-transplant blood sample was not available, swab of buccal mucosa was used for pre-transplant STR profile. A standard set of primers for STR markers were used and the amplified DNA was resolved by gel electrophoresis and stained with silver nitrate. The percentage of donor origin DNA was estimated by densitometer., Results: Out of 84 patients, 52 (62%) were males, while 32 (38%) were females. In patients of beta-thalassaemia major, 31 (62%) developed mixed donor chimerism (MC), 13 (26%) developed complete donor chimerism (CC) and 6 (12%) had graft failure. In aplastic anaemia, 17 patients (50%) achieved MC, 13 (38.2%) had CC and 4 (11.8%) developed graft failure. The combined frequency of mixed donor chimerism for both the diseases was 58.3%. D3S1358 was the most informative STR marker in these patients., Conclusion: Majority of the studied patients developed mixed donor chimerism following bone marrow transplantation, whereas only a minor percentage of the patients had graft failure. Analysis of D3S1358 was the most informative in assessing donor chimerism in patients who underwent BMT.

The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance) were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM) and specific (concentration of unexpected amplicons <2 nM) amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites) in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.

Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

The polymorphism of short tandem repeats (STRs) is commonly used for human identity testing. Many commercial kits are available for this purpose, in which multiplex PCR for 10-16 STRs is usually used. For optimal results, the kits recommend rather limited conditions, which not all forensic samples can satisfy. We increased the efficiency of several commercial kits by modifying the components and reaction parameters (primer concentration and cycle number, and annealing and extension time). To simulate low copy number, we used 1:10 and 1:100 diluted samples compared to the recommended concentration. A change in cycle number, annealing and extension time, and primer concentration showed various results. Fine-tuning of PCR conditions by combining the described changes, decreasing the primer concentration, and increasing the annealing and extension time together with increasing the cycling number dramatically increased the efficiency of the reaction, even in low-copy-number simulated samples. Detailed information for several kit components or kits with different components targeting difficult sample conditions, including low copy number, would be of great help in forensics.

The purpose of this work is to define rare variants of cystic fibrosis (CF) that are potential sources of error and can confound molecular genetic testing methods. We performed routine, clinical CF mutation screening using a laboratory-developed test and the oligonucleotide ligation assay reagents from Abbott/Celera. In this report, we describe 11 unique allele drop outs [3849 + 10kb C>T (NM&#95;000492.2:c.3718-2477C>T), V520F (c.1558G>T), 1078delT (c.948delT), A455E (c.1364C>A), R347P (c.1040G>C), 2184delA (c.2052delA), W1282X (c.3846G>A), R117H (c.350G>A), G85E (c.254G>A), 621 + 1G>T (c.489 + 1G>T), and 2789 + 5G>A (c.2657 + 5G>A)] observed with this platform. The allele drop outs account for less than 0.01% of all results reported in our laboratory. Both the recognition and enumeration of such variants along with clinical information in CF testing is valuable in avoiding false-positive and false-negative results.

Sulfide production by sulfate-reducing bacteria (SRB) is a major concern for the petroleum industry since it is toxic and corrosive, and causes plugging due to the formation of insoluble iron sulfides (reservoir souring). In this study, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) using two sets of primers based on the 16S rRNA gene and on the aps gene (adenosine-5-phosphosulfate reductase) was used to track changes in the total bacterial and SRB communities, respectively, present in the water-oil tank system on an offshore platform in Brazil in which nitrate treatment was applied for 2 months (15 nitrate injections). PCR-DGGE analysis of the total bacterial community showed the existence of a dominant population in the water-oil tank, and that the appearance and/or the increase of intensity of some bands in the gels were not permanently affected by the introduction of nitrate. On the other hand, the SRB community was stimulated following nitrate treatment. Moreover, sulfide production did not exceed the permissible exposure limit in the water-oil separation tank studied treated with nitrate. Therefore, controlling sulfide production by treating the produced water tank with nitrate could reduce the quantity of chemical biocides required to control microbial activities.

This paper presents a micro polymerase chain reaction (PCR) chip for the DNA-based diagnosis of microorganism genes and the detection of their corresponding antibiotic-resistant genes. The micro PCR chip comprises cheap biocompatible soda-lime glass substrates with integrated thin-film platinum resistors as heating/sensing elements, and is fabricated using micro-electro-mechanical-system (MEMS) techniques in a reliable batch-fabrication process. The heating and temperature sensing elements are made of the same material and are located inside the reaction chamber in order to ensure a uniform temperature distribution. This study performs the detection of several genes associated with upper respiratory tract infection microorganisms, i.e. Streptococcus pneumoniae, Haemopilus influenze, Staphylococcu aureus, Streptococcus pyogenes, and Neisseria meningitides, together with their corresponding antibiotic-resistant genes. The lower thermal inertia of the proposed micro PCR chip relative to conventional bench-top PCR systems enables a more rapid detection operation with reduced sample and reagent consumption. The experimental data reveal that the high heating and cooling rates of the system (20 and 10 degrees C/s, respectively) permit successful DNA amplification within 15 min. The micro PCR chip is also capable of performing multiple DNA amplification, i.e. the simultaneous duplication of multiple genes under different conditions in separate reaction wells. Compared with the large-scale PCR system, it is greatly advantageous for fast diagnosis of multiple infectious diseases. Multiplex PCR amplification of two DNA segments in the same well is also feasible using the proposed micro device. The developed micro PCR chip provides a crucial tool for genetic analysis, molecular biology, infectious disease detection, and many other biomedical applications.

The present communication presents a new triplex PCR co-amplifying three loci (D3S1358, D8S1179 and D18S51) recommended for STR typing by the European Network of Forensic Science Institutes (ENFSI). Twenty-two different primers were tested to optimise the PCR. Four of the six primer sequences finally chosen were self selected, the fifth was a published one and the sixth derived from a commercially available multiplex kit. Using this PCR-setup, even minimum amounts of genomic DNA are sufficient to analyse the STR loci D3S1358, D8S1179 and D18S51 in parallel. Especially in forensic casework, where DNA is mostly limited and often contaminated with enzyme inhibitors, this new PCR proved to be very advantageous. To demonstrate the reliability, buccal swabs from 2874 persons were typed not only with the new triplex PCR but also with a commercially available multiplex kit.

To facilitate identification of ehrlichial pathogens, we developed a new technique based on fingerprints resulting from repetitive element polymerase chain reaction (rep-PCR). This technique uses consensus tRNA primers to generate amplification products that reflect distance polymorphisms between adjacent tRNA genes. Species-specific fingerprint patterns were obtained for seven Ehrlichia spp., as well as the unnamed causative agent of human granulocytotropic ehrlichiosis. Bands ranged in size from approximately 50 to 1,000 base pairs. Banding patterns varied depending on dilution of template DNA, with lower dilutions giving more complex banding patterns. These preliminary data indicate that repetitive-sequence-based PCR appears to be a useful technique for identifying ehrlichial organisms to the species, and perhaps the strain level. Compared with other conventional molecular-biologic methods, rep-PCR offers the advantages of ease of performance and rapid availability of results.

Objective: To develop a sensitive method for genetic diagnosis using capillary electrophoresis with laser-induced fluorescence., Design: Using male DNA diluted with female DNA as an example, ZFY gene from Y chromosome was amplified specifically by polymerase chain reaction (PCR) with fluorescence-labeled primers and detected by capillary electrophoresis with laser-induced fluorescence., Setting: Laser operating laboratory., Participants: Human male and female DNA were extracted from healthy human male and female subjects., Interventions: None., Main Outcome Measures: The concentration of human DNA was determined by using a spectrophotometer at an absorbance of 260 nm., Result: Deoxyribonucleic acid fragments amplified from a single copy of ZFY gene were detected by capillary electrophoresis with laser-induced fluorescence after 35 cycles of PCR amplification., Conclusion: This method is potentially applicable for rapid and sensitive detection of fetal Y chromosome DNA sequence in maternal circulation and of single-cell DNA diagnosis.

The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T-NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T-cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.

Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

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Background: Familial chylomicronemia syndrome (FCS) is a rare monogenic form of severe hypertriglyceridemia, caused by mutations in genes involved in triglyceride metabolism. Herein, we report the case of a Korean family with familial chylomicronemia syndrome caused by compound heterozygous deletions of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). Case presentation: A 4-year-old boy was referred for the evaluation of severe hypertriglyceridemia (3734 mg/dL) that was incidentally detected 4 months prior. His elder brother also demonstrated an elevated triglyceride level of 2133 mg/dL at the age of 9. Lipoprotein electrophoresis revealed the presence of chylomicrons, an increase in the proportion of pre-beta lipoproteins, and low serum lipoprotein lipase levels. The patient's parents and first elder brother had stable lipid profiles. For suspected FCS, genetic testing was performed using the next-generation sequencing-based analysis of 31 lipid metabolism-associated genes, which revealed no pathogenic variants. However, copy number variant screening using sequencing depth information suggested large heterozygous deletion encompassing all the coding exons of GPIHBP1. A real-time quantitative polymerase chain reaction was performed to validate the deletion site. The results showed that the siblings had two heterozygous copy number variants consisting of the whole gene and an exon 4 deletion, each inherited from their parents. During the follow-up period of 17 months, the patient did not develop pancreatitis, following dietary intervention. Conclusion: These siblings' case of familial chylomicronemia syndrome caused by rare GPIHBP1 deletions highlight the implementation of copy number variants—beyond next-generation sequencing—as an important consideration in diagnosis. Accurate genetic diagnosis is necessary to establish the etiology of severe hypertriglyceridemia, which increases the risk of pancreatitis. [ABSTRACT FROM AUTHOR]

Gene expression analysis using the qPCR method requires an oligonucleotide pair to prime the amplification process. Supporting the widely used qPCR method, variety of qPCR reagents and primer options are available. This infers to the importance of in silico and laboratory experimental validation approach considering SYBR Green optimum annealing temperature to validate the most suitable primer for prior use. Several genes are suspected to be involved in the BM-MSCs migration and differentiation into Cancer-associated Fibroblast Cells in Rattus norvegicus. This article aims to provide in silico analysis with the case of the suspected genes namely actin alpha-2 smooth muscle (ACTA2), fibroblast activation protein (FAP), hypoxanthine phosphoribosyltransferase-1 (HPRT1), platelet-derived growth factor subunit B (PDGFB), phosphoinositide-3-kinase regulatory subunit-1 (PIK3R1), and vascular cell adhesion molecule-1 (VCAM1) qPCR primer with qPCR and electrophoresis validation. The procedure used in this approach was in silico analysis of primer from published articles and newly designed primer. The analysis was done with Primer-BLAST for gene specificity, Primer-Dimer, OligoCalc for hairpin formation, BLAST Nucleotide for identical sequence screening, and Clustal Omega for product length validation. Experimental validation was done using qPCR for optimal annealing temperature, priming ability, amplicon specificity, and electrophoresis for product length validation. This assessment resulted in in silico and laboratory experimental validation of ACTA2, FAP, HPRT1, PDGFB, PIK3R1, and VCAM1 primer pairs producing suitable amplicon for qPCR using Rattus norvegicus cDNA with SYBR annealing temperature range of 60-65° C with three mM MgCl2. The primer pairs can be used for further qPCR analysis under similar conditions and the procedure stated can be used as starting point for qPCR Primer preparation accounting for fluorophore optimum annealing temperature. [ABSTRACT FROM AUTHOR]

Studies on coral genetics have been widely used recently for varied research purposes, including to solve the taxonomical challenge or to understand the physiological-ecological aspects of corals towards climate change. While the information provided by coral genome is crucial for those works, isolation of coral genome is still a difficult preliminary step due to coral's nature. This study aimed to evaluate and to suggest the best application of among four established methods for DNA extraction from scleractinian coral, i.e., Guanidine hydrochloride (GuHCl), Chelex chelating resin, Guanidinium thiocyanate-phenol-chloroform (AGPC), and spin-column methods. Coral samples were grounded and submitted to each extraction method's protocol. Final extracts were checked for their quality and quantity. The final extracts feasibility were tested through PCR (Polymerase Chain Reaction), followed by electrophoresis for amplicon quality screening. The scoring was done for assessment aspects: time efficiency, successful extraction process, extracted DNA quantity and quality, final extract properness, and cost effort per sample. The highest DNA concentration was obtained by spin-column methods, reaching up to 724.31 ng/µl, while other methods can only provide between 264.94±36.453 to 378.19±0 ng/µl of DNA. Among all method tested, the spin-column was able to provide reproducible and feasible quality of final extracts for the amplification. Other methods were failed to provide such consistent and proper results. The results suggesthat the spin-column is the most appropriate method for DNA isolation from scleractinian specimen.

Based on the analysis of patients with Peutz-Jeghers syndrome (PJS), Serine threonine kinase11 (STK11) is known as a tumor suppressor gene, which is involved in cell polarization, regulation of apoptosis, and DNA damage response. In this case report study, we examined STK11 gene sequencing in a 42-year-old woman with mucocuta neous pigmentation and positive family history. Endoscopy and colonoscopy showed >1000 polyps throughout the stomach/colon (PJ-type hamartomas). The larger polyp in the stomach was resected and the small bowel imaging detected multiple jejunum/ileum small polyps. The data released from the sequencing results revealed five alterations in exons 1 to 5. The major mutation in stop codon was reported as converted to the amino acid tryptophan (TRP) to tyrosine (TER). The TGG codon was converted to TAG by mutation. Finally, another novel mutation in STK11 stop codon as a 'de novo' variant was seen. It is predicted that stop codon mutations make the affected person susceptible to developing colorectal cancer. [ABSTRACT FROM AUTHOR]

Background and Aim: Entamoeba histolytica and Giardia intestinalis are two common parasites associated with gastrointestinal infections and diarrheal diseases worldwide. This study investigated the prevalence, molecular characterization, and phylogenetic relationships of E. histolytica and G. intestinalis among patients with diarrhea and intestinal disorders in hospitals across Iraq. Materials and Methods: Stool samples were collected from individuals presenting with gastrointestinal symptoms, and a microscopic examination was performed to screen for the presence of E. histolytica and G. intestinalis. DNA was extracted from positive samples, and PCR amplification targeting the 16S rRNA gene for E. histolytica and the triose phosphate isomerase (TPI) gene for G. intestinalis was carried out. The resulting PCR products were subjected to agarose gel electrophoresis, purified, and sequenced. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Tamura-Nei model, and bootstrap analysis was performed. Results: Among the studied samples, 27.8% (n=85) were positive for E. histolytica, while 19.6% (n=60) were positive for G. intestinalis. Sequence analysis confirmed the presence of E. histolytica and G. intestinalis by matching the obtained sequences with those available in the NCBI gene bank. The phylogenetic analysis revealed the genetic diversity and relatedness of the E. histolytica and G. intestinalis isolates. Clustering patterns specific to the Iraqi population were observed, suggesting potential regional transmission dynamics. Conclusion: This study provides valuable insights into the prevalence, molecular characterization, and phylogenetic relationships of E. histolytica and G. intestinalis in Iraqi patients with diarrhea and intestinal disorders. [ABSTRACT FROM AUTHOR]

Background and Aim: Oral infections are common among people of any age and can trigger systemic inflammation. The microbiota is a diverse group of microorganisms that play important roles in metabolism, immune function, and homeostasis. Oral microbiota in human atherosclerotic plaques has been identified using various techniques. Therefore, the focus of this study was to determine the correlation between metabolic parameters and oral microbiota composition in patients with atherosclerosis using Denaturing Gradient Gel Electrophoresis (DGGE) assays. Materials and Methods: In this case-control study, saliva samples were collected from 139 patients with atherosclerosis and healthy individuals from Imam Ali Cardiovascular Hospital, Kermanshah, Iran. After DNA extraction, PCR products were examined and evaluated using DGGE assays. Results: The study included 89 (36%) patients with a history of atherosclerosis and 50 (36%) healthy individuals. There was a significant relationship between the mean total cholesterol, Low-Density Lipoprotein (LDL), Fasting Blood Sugar (FBS), and Blood Urea Nitrogen (BUN) in the two groups. However, there was no significant difference in the mean high-density lipoprotein (HDL) and Triglyceride levels between the study groups. Conclusion: Our results showed a relationship between metabolic parameters and oral microbiota composition in patients with atherosclerosis. Additionally, our results indicated that the DGGE assay is a useful method for diagnosing and comparing the oral microbiota of people with atherosclerosis and healthy individuals. Therefore, further examination of the oral microbiota is necessary to determine its potential as a biomarker for atherosclerosis. [ABSTRACT FROM AUTHOR]

Objective Our aim was to establish a chemiluminescence method for detecting anti-transmembrane protein (p7) antibody in the serum of patients with hepatitis C virus (HCV) infection. Methods The p7 gene was amplified by polymerase chain reaction using the plasmid PUC-p7 containing the p7 nucleic acid sequence of the HCV 1b genotype as the template, and recombinant plasmid pGEX-KG-p7 was constructed. After p7 fusion, the protein was induced and expressed in the prokaryote, extracted, and purified; the anti-p7 antibody detection kit was prepared, and its efficacy was evaluated. Results The plasmid pGEX-KG-p7 was constructed correctly, and p7 fusion protein was obtained. The methodological indexes of the kit, the precision test, blank limit and detection limit, etc, met the requirements. The positive rate of serum anti-p7 antibody in 45 patients with HCV infection was 20%. Conclusions The kit can be used in screening diagnosis, condition monitoring, prognosis, and disease mechanism and epidemiological study of HCV infection. The p7 protein has immune response in HCV-infected patients. [ABSTRACT FROM AUTHOR]

In the context of solving the problem of obtaining high quality dairy products from livestock, the issue of determining the type of beta-casein (A1 and A2) in the protein fraction of milk becomes essential. Purpose - to analyse the use of ACRS-PCR methods for differentiation of A¹ and A² alleles of bovine beta-casein locus. Genotyping features were analysed using the artificially created restriction site polymerase chain reaction method utilising TaqI and Ddel restriction endonucleases. The electrophoretic distribution of DNA fragments in agarose gels of various concentrations was used to analyse restriction patterns. Based on the results of bioinformatic analysis of the nucleotide reference sequences of the experimental fragment of the beta-casein gene, it was found that the primer system for the ACRS-PCR DdeI method is characterised by higher parameters of flanking efficiency of the target DNA site compared to the ACRS-PCR TaqI system due to significantly greater effectiveness of hybridisation of oligonucleotides on the target DNA. Based on the results of laboratory tests of both methods, it is proposed to use an additional procedure for analysing the fluorescence intensity of individual elements of restriction patterns, which allows reducing the number of false genotyping that occurs in both cases (based on the results of using both methods) due to the appearance of non-specific amplification/restriction fragments within the size of target restrictions. The application of the ACRS-PCR DdeI method provides more differentiated patterns of the corresponding genotypes in agarose gel compared to the ACRS-PCR TaqI method, but leads to higher material costs for conducting research. These disadvantages of using primer systems for ACRS-PCR of the beta-casein locus determine the relevance of developing alternative methods for typing A¹ and A² alleles which include allele-specific PCR. The use of results is promising for solving the problems of genotyping cattle individuals of different breeds by A ¹ and A² alleles of the beta-casein locus [ABSTRACT FROM AUTHOR]

Introduction: Despite careful control of environmental factors, there is a wide variety of interpersonal muscle-induced muscle damage. Genetic changes can also be important in determining susceptibility to injury. Weightlifting is one of the most successful Iranian sports teams in Olympics; so, the aim of this study was the investigation of the relationship between ACE I/D gene polymorphism rs4646994 and incidence and severity of sport injuries in Iranian male elite weightlifters. Materials and Methods: The study was cross-sectional research that conducted in 2022. The statistical population consisted of male elite weightlifters from Isfahan consisting of 31 athletes who all were investigated as sample. A questionnaire was used to collect the injuries data. Saliva samples were taken from all subjects and DNA was extracted and genotyped by using real-time polymerase chain reaction (real time-PCR). Chi-square and regression tests were used to analyze the data. Significance level was also considered at P <0.05. Results: There was a significant difference in the incidence of injuries in different genotypes. ID genotype (59.4%) had the highest incidence of injury in compare with DD (23.2%) and II (17.4%) genotypes (χ2 = 21.47, P = 0.001). Also, there was a significant difference in the severity of injuries in different genotypes (χ2 =15.59, P = 0.049); but there was no relationship between genotype and incidence and the severity of sports injuries in selected Iranian elite male weightlifters (P= 0.715). Conclusion: The results showed differences in the incidence and severity of sport injuries in different genotypes, hence it would be important in the prediction injury risk in weightlifters. [ABSTRACT FROM AUTHOR]

Background: Aging is associated with impaired renal function and structural alterations. Oxidative stress plays a vital role in renal senescence and damage. Sirtuin 1 (SIRT1) is thought to protect cells from oxidative stress through nuclear factor erythroid 2-related factor 2 (NRF2). Ellagic acid (EA), a natural antioxidant, has been demonstrated to have renoprotective roles in vitro and in vivo. This study investigated if SIRT1 and NRF2 mediate the protective effects of EA in aged kidneys. Methods: Male Wistar rats were divided into three groups including young (4 months), old, and old + EA (25 months). Young and old groups received EA solvent, while the old + EA group was treated with EA (30 mg/kg) by gavage for 30 days. Then, the level of renal oxidative stress, SIRT1 and NRF2 expression, kidney function parameters, and histopathological indices were measured. Results: Treatment with EA significantly increased the level of antioxidant enzymes and reduced malondialdehyde concentration (P < 0.01). Moreover, EA administration remarkably upregulated mRNA and protein levels of SIRT1 and NRF2 as well as deacetylated NRF2 protein (P < 0.05). Additionally, EA treated rats improved kidney function and histopathological scores (P < 0.05). Conclusions: These findings suggest that ellagic acid exerts protective effects on aged kidneys by activating SIRT1 and NRF2 signaling. [ABSTRACT FROM AUTHOR]

Although breast cancer screening assays exist, many are inaccessible and have high turnaround times, leaving a significant need for better alternatives. Hypermethylation of tumor suppressor genes is a common epigenetic marker of breast cancer. Methylation tends to occur most frequently in the promoter and first exon regions of genes. Preliminary screening tests are crucial for informing patients whether they should pursue more involved testing. We selected RASSF1, previously demonstrated to be aberrantly methylated in liquid biopsies from breast cancer patients, as our gene of interest. Using CoBRA as our method for methylation quantification, we designed unique primer sets that amplify a portion of the CpG island spanning the 5′ end of the RASSF1 first exon. We integrated the CoBRA approach with a microfluidics-based electrophoresis quantification system (LabChip) and optimized the assay such that insightful results could be obtained without post-PCR purification or concentration, two steps traditionally included in CoBRA assays. Circumventing these steps resulted in a decreased turnaround time and mitigated the laboratory machinery and reagent requirements. Our streamlined technique has an estimated limit of detection of 9.1 ng/μL of input DNA and was able to quantify methylation with an average error of 4.3%. [ABSTRACT FROM AUTHOR]

Children carrying the minor allele 'A' at the fat mass and obesity-associated protein (FTO) gene have higher obesity prevalence. We examined the link between FTO rs9939609 polymorphism and plasma adiponectin and the mediating role of body adiposity, in a cross-sectional study comprising 323 children aged 6–11 years. Adiponectin and FTO genotypes were assessed using a commercial kit and a real-time polymerase chain reaction with high-resolution melting analysis, respectively. Body adiposity included body mass index z-score, body fat percentage and waist-to-hip ratio. To investigate adiponectin (outcome) associations with FTO and adiposity, linear regressions were implemented in additive models and across genotype categories, adjusting for sex, age and Tanner's stage. Using mediation analysis, we determined the proportion of the association adiponectin-FTO mediated by body adiposity. Lower adiponectin concentrations were associated with one additional risk allele (βadditive = −0.075 log-μg/mL [−0.124; −0.025]), a homozygous risk genotype (βAA/TT = −0.150 [−0.253; −0.048]) and a higher body mass index z-score (β = −0.130 [−0.176; −0.085]). Similar results were obtained for body fat percentage and waist-to-hip ratio. Body adiposity may mediate up to 29.8% of the FTO-adiponectin association. In conclusion, FTO rs9939609-related differences in body adiposity may partially explain lower adiponectin concentrations. Further studies need to disentangle the biological pathways independent from body adiposity. [ABSTRACT FROM AUTHOR]

Background: The pro-inflammatory cytokine, interleukin 16 (IL-16), has been shown to be secreted in low levels in knee osteoarthritis (KOA). The aim of the study was to examine the relationship between IL-16 polymorphisms and the risk of KOA in the Egyptian population, as well as the clinical and radiographic severity of KOA. Results: IL16 rs11556218 thymidine triphosphate (T) T G (guanosine triphosphate), GG, TG + GG genotypes, and G allele (odd ratio (OR) = 0.315; 95% confidence interval (CI) = 0.191–0.518; P < 0.001; OR = 0.363; 95% CI = 0.162–0.815, P = 0.014; OR = 0.323; 95% CI = 0.202–0.519, P < 0.001; OR = 0.480; 95% CI = 0.338–0.683, P < 0.001 respectively); rs4778889 cytidine triphosphate (C) T,CC, TC + CC genotypes, and C allele (OR = 0.519, 95% CI = 0.319–0.844, P = 0.008; OR = 0.309, 95% CI = 0.105–0.916, P = 0.034; OR = 0.485, 95% CI = 0.304–0.775, P = 0.002; OR = 0.537, 95% CI = 0.365–0.791, P = 0.001 respectively); and rs4072111 CT, TT, CT + TT genotypes, and T allele (OR = 0.537, 95% CI = 0.323–0.893, P = 0.017, OR = 0.316, 95% CI = 0.096–0.843, P = 0.049, OR = 0.502, 95% CI = 0.309–0.816, P = 0.005; OR = 0.534, 95% CI = 0.353–0.809, P = 0.004 respectively) were associated with a decreased KOA risk, and they were significantly associated with decreased the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and the Kellgren-Lawrence (K/L) scores. Neither IL-16 serum levels nor IL-16 polymorphisms were associated with the susceptibility to KOA. Low KOA risk was associated with the haplotypes GTC and TCT. Conclusion: There was no correlation between serum IL-16 levels and KOA susceptibility or IL-16 polymorphisms. GTC and TCT haplotypes were associated with low KOA risk. The variant alleles rs11556218GG, TG + GG; rs4778889 CC, TC + CC; and rs4072111 TT, CT + TT were associated with a reduced risk of KOA. [ABSTRACT FROM AUTHOR]

Background and Aim: Otitis media (OM) or middle ear inflammation is an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect nan1 and tox A genes of P. aeruginosa isolated from chronic OM patients. Materials and Methods: One hundred thirty OM patients were recruited from Al-Sader medical city and Al-Hakim general hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 115 samples (88.46%) give a positive result, while 15 samples (11.54%) are negative for culture bacteria. Only 49 (42.76 %) of the 115 clinical specimens showed positive tests for P. aeruginosa. However, the toxA gene is found in 67.3% of P. aeruginosa isolates, while the nan1 gene is found in 38.77 % of the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of OM infection in Iraqi patients. Most bacterial isolates show the presence of nan1 and tox A genes, with a high prevalence of tox A than nan1. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics. [ABSTRACT FROM AUTHOR]

Advancements in Polymerase Chain Reaction (PCR) technology and other techniques like Deoxyribonucleic acid (DNA) signal and target amplification have become key procedures in molecular diagnostics. PCR enables the synthesis of nucleic acids in vitro through which a DNA segment can be specifically replicated in a semiconservative way that sets forth deletion and mutation analysis. Multiplex PCR (M-PCR) is beneficial over standard and long PCR as this can amplify more than one locus using the respective primer sets. In harmony with this, the present study aimed to optimize M-PCR followed by its chemistry and condition to screen Duchenne Muscular Dystrophy (DMD) [OMIM #310200] and Becker Muscular Dystrophy (BMD) [OMIM #300376]. Muscular Dystrophies (MDs) are a broad group of hereditary, progressive, and degenerative disorders of muscles. X-linked recessive D/BMD are caused by mutation/s in the dystrophin gene [OMIM #300377] that encodes for dystrophin protein [UniProt#P11532]. As dystrophin is the human metagene with 79 exons, mutational analysis is very challenging. Chamberlain set (10 plex), Beggs set (9 Plex), and Kunkel set (7 Plex) is used for many years to diagnose this condition. However, in this study, Beggs set is customized with 13 exons to screen DMD gene mutation in a single reaction. Optimization of M-PCR was designed with many physicochemical parameters. According to the literature and after many appraisals the present study demonstrated the most sufficient concentration of various chemical components and optimal cycling conditions to optimize the modified Beggs set (13 Plex). 50 µL PCR reaction includes primer(s) (0.3-0.5 µM each), dNTP mixture (160 µM each), Dream Taq buffer (1X), Taq DNA polymerase (6U/50 µL), DNA template (250 ng/50 µL), BSA (0.4 µg/µL), and MgCl2 (1.4 mM). To get the most effective results cyclic conditions obtained were 10 min initial denaturation at 94°C, 62°C annealing temperature, and 35 PCR cycles at 72°C extending temperature. Consequently, the study successfully formulated a less expensive and simple approach for >3000 bp that was used to screen D/BMD. Finally, a developed M-PCR mix with a unique combination of specificity and sensitivity coupled with great flexibility has led to a true revolution in molecular diagnostics. [ABSTRACT FROM AUTHOR]

Background: Non-Syndromic Cleft Lip with or without cleft Palate (NSCL/P) is a common developmental disorder of the head and neck with a multifactorial etiology. The current study aimed to evaluate the potential association of PTCH1 (rs10512248) and RAD54B (rs12681366) polymorphisms with NSCL/P in the Northeast Iranian population. Methods: In the present study, blood samples were taken from 122 subjects with NSCL/P and 161 healthy controls. Polymerase Chain Reaction (PCR) followed by Restriction Fragment Length Polymorphism (RFLP) were used to conduct genotyping of single-nucleotide polymorphisms. Results: Although differences were observed between cases and controls in rs10512248 and rs12681366, our data did not support a significant association of these polymorphisms with NSCL/P in our population. Conclusion: Our findings suggest that polymorphisms of rs10512248 and rs12681366 may not be potential risk factors for NSCL/P in the Northeast Iranian population due to the multifactorial and multiethnicity characteristics of some genes. [ABSTRACT FROM AUTHOR]

We present the Pneumocystis pneumonia case of a 64-year-old man with no remarkable history except for hypertension, who had not undergone any treatment other than surgery. On postoperative day 7, high-resolution computed tomography findings revealed multifocal ground-glass opacifications with interlobular septal thickening in both lungs; therefore, atypical pneumonia was suspected. Polymerase chain reaction (PCR) test performed after bronchoalveolar lavage was positive for Pneumocystis jirovecii (P. jirovecii). Based on the PCR results, a final diagnosis of Pneumocystis pneumonia (PCP) was made. After treatment, he improved and was discharged. This is a unique case of PCP diagnosis in a non-immunocompromised patient, with no remarkable history except for hypertension, who had not undergone any treatment other than surgery for cancer. Thus, it is necessary to consider additional risk factors for PCP and timing of preventive treatment. [ABSTRACT FROM AUTHOR]

In most procedures that involve gene cloning, after the amplification of a target gene by PCR or by Real-time PCR, the purification of the trapped gene on agarose gel is a crucial stage. There are various methods for extracting genes from agarose gel by removing other contaminants. We isolated the amplified PqHMGR gene (derived from Ginseng (Panax quinquefolius)) from agarose gel by a quasi-electrophoresis device (similar to electro-elution technique). Moreover, the efficiency of this new approach was compared with that of the commercial kit 'Silica Bead DNA Gel Extraction' (Thermo Scientific American Company). Ligation to the PTG- 19 plasmid and cloning in E. coli bacteria were also done. The results showed successful isolations of targeted DNA, along with a high efficiency in producing recombinant DNA and in concluding a successful cloning procedure through this new device. The invented method provided a better purification ability than the commercial kit, but because of using the TAE 1X buffer as the purified gene storage solution, the plasmid and bacterial transformation rates were slower than the commercial kit method. It was found that using the new method for the purification of nucleotide sequences by electrophoresis and electrophoresis buffer is feasible, and that these purified fragments can be applied in cloning and sequencing. Using the TAE 1X buffer instead of distilled water did not cause problems in gene binding to PTG-19 plasmid. It also allowed a successful transformation of E. coli bacteria by the modified plasmid. Nonetheless, using TAE 1X buffer reduced the modification rate of the PTG-19 plasmid and decreased the rate of E. coli transformation by the modified plasmid. [ABSTRACT FROM AUTHOR]

Effluents discharged by poultry meat industries are heavily polluted with raw materials, such as fat, blood residues, and proteins. Thus, untreated effluents directly discharged into the environment may constitute a public health threat. This study aims to evaluate the bacterial diversity of three water qualities: industrial poultry wastewater (PWW), tap water (TW), and PWW diluted with TW (50 : 50) (V/V) (TWPWW) by the combination of culture-independent and culture-dependent approaches. The total bacterial DNA was extracted using phenol/chloroform method. The hypervariable 16S rRNA region V3-V5 was amplified by PCR using universal primers. The amplicons were separated by vertical electrophoresis on a polyacrylamide gel of increasing denaturing gradient according to their richness in GC bases. Selected bands were reamplified and sequenced. Pure isolated bacteria from nutrient agar medium were characterized according to their morphological and biochemical characteristics. Genomic DNA from pure strains was extracted by boiling method, and a molecular amplification of the 16S–23S ITS region of the 16S rRNA gene was performed using the universal primers. Selected isolates were identified by sequencing. Results showed a high bacterial load and diversity in PWW in comparison with TW and TWPWW. A collection of 44 strains was obtained, and 25 of them were identified by sequencing. Proteobacteria represented 76% of isolated bacteria Gamma-Proteobacteria was the predominate isolate (68%). Other isolates were Firmicutes (8%), Bacteroidetes (12%), and Actinobacteria (8%). These isolates belong to different genera, namely, Pseudomonas, Acinetobacter, Proteus, Empedobacter, Corynebacterium, Enterobacter, Comamonas, Frondibacter, Leclercia, Staphylococcus, Atlantibacter, Klebsiella, and Microbacterium. [ABSTRACT FROM AUTHOR]

Background. Gastric cancer (GC) is a malignant tumor of the gastrointestinal system. Since the early symptoms of GC are not obvious and lack efficient diagnostic markers, it is urgent to find new diagnostic markers with good sensitivity and specificity. tRNA-derived small RNAs (tsRNAs) are an emerging class of small noncoding RNAs with good abundance in body fluids. We aim to find new tsRNAs as biomarkers for GC diagnosis. Methods. High-throughput sequencing was used to identify differentially expressed tsRNAs in GC tissues, and quantitative real-time PCR was used to detect the expression level of tRF-17-WS7K092. Agarose gel electrophoresis and Sanger sequencing were performed to verify the characteristics of tRF-17-WS7K092. The diagnostic efficacy of tRF-17-WS7K092 was analyzed by the receiver operating characteristic curve. Results. In this study, the expression levels of tRF-17-WS7K092 were significantly increased in GC tissues, cells, and serum. After GC surgery, the expression level of serum tRF-17-WS7K092 decreased, and its high expression was associated with low survival rates. In addition, the expression level of serum tRF-17-WS7K092 was correlated with the T stage, TNM stage, lymph node metastasis, and nerve/vascular invasion and could distinguish GC patients from gastritis patients and healthy donors as well. Conclusions. The expression of serum tRF-17-WS7K092 was significantly increased in GC and decreased after GC surgery, suggesting that serum tRF-17-WS7K092 may serve as a promising biomarker for the diagnostic and prognostic monitoring of GC. [ABSTRACT FROM AUTHOR]