Polymerase Chain Reaction Technology Research Articles

Simultaneous Detection of Eight Dairy-Derived Components Using Double-Tube Multiplex qPCR Based TaqMan Probe.

The authentication of milk and dairy products has great significance for food fraud. The present investigation entailed the development of a novel method that amalgamates the double-tube approach with multiplex real-time polymerase chain reaction (PCR) technology, incorporating TaqMan probes, to facilitate the high-throughput screening and detection of animal-derived constituents within milk and dairy products. Eight dairy-derived animal-specific primers and probes were designed for the mitochondrial cytochrome b (Cytb) gene of eight dairy products, including cow, buffalo, yak, goat, sheep, horse, donkey, and camel. Through the developed double-tube detection assays, the above eight targets could be simultaneously identified with a detection limit of 0.00128-0.0064 ng/μL. The multiplex qPCR assay was effectively validated using simulated adulterated samples with different mixing ratios and demonstrated a detection limit of 0.1%. Upon analysis of 54 commercially available dairy products, a mislabeling rate of 33% was revealed. This method affords an efficacious means of detecting dairy product ingredients, thereby offering robust technical backing for market oversight and regulatory enforcement of milk and dairy products.

Molecular diagnosis of E.coliO157:H7 Which Isolated from Children with Diarrhea by using Multiplex PCR

A total of 96 stool samples were collected from children with bloody diarrhea from two hospitals in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identifed by using morphological diagnostic methods, Samples were cultured on liquid enrichment medium, incubated at 37C? for 24 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 32 non-sorbitol fermenting bacterial isolates were obtained of which 11 were identified as Escherichia coli by using traditional biochemical tests and API20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates . Four special biochemical tests were done for serotype O157:H7 differentiation from other NSF bacteria. Only 3 isolates belonging to the serotype O157:H7 were obtained . Latex agglutination test for O157 and H7 showed that the 3 isolates gave positive results with both tests. The Bacterial isolates were identifed by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated By using specific primers in MPCR . The result showed that one E. coli O157:H7 isolates contain all 4 genes , other isolates contain 3 genes: Stx2, hlyA & eaeA.

Development and validation of a droplet digital PCR method for quantifying lentiviral vector infectious titer

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

Distribution characteristics of integrons and correlation analysis of antibiotic resistance in urine isolated Enterobacter cloacae.

This study aims to understand the distribution of integrons among Enterobacter cloacae isolated from clinical urine specimens in our hospital, as well as the molecular characteristics of the variable region resistance gene cassette of integron-positive strains and its relationship with drug resistance. We collected a total of 80 strains of Enterobacter cloacae isolated from urine specimens of hospitalized patients in our hospital between August 2019 and July 2023, and conducted drug sensitivity testing on them. Polymerase Chain Reaction (PCR) technology was employed to screen these strains for Class 1, 2, and 3 integrons. Following this, the promoter and variable regions of integron-positive strains were amplified and sequenced. Additionally, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) was utilized for homology analysis of integron-positive strains. Among the 80 clinical strains, Class 1 integrons were detected in 31 (38.8%) strains, and the following resistance gene cassettes were identified: aadA2, aadA1, aadB, aac(6'), and catB8. Three types of variable region promoters were observed: PcS (4 strains), PcW (7 strains), and PcH1 (17 strains), with consistently inactive downstream P2 promoters. Additionally, Class 2 integrons were detected in 5 (6.3%) strains, carrying the variable region resistance gene cassette dfrA1-sat2-aadA1. The promoters for Class 2 integrons were uniformly of the Pc2D-Pc2A-Pc2B-Pc2C type. No Class 3 integrons were detected. The strains containing integrons showed significantly higher resistance rates to ciprofloxacin, compound sulfamethoxazole, levofloxacin, gentamicin, amikacin, and tobramycin compared to those without integrons (P<0.05). 35 strains of Enterobacter cloacae carrying integrons are primarily classified into three genotypes: A, B, and C. These genotypes are mainly distributed in the urology department and Intensive Care Unit (ICU). The distribution of variable region gene boxes and promoter types is relatively concentrated in the same genotype. Our study confirmed that Enterobacter cloacae isolated from urine samples predominantly carries Class 1 integrons with an extended array of antibiotic-resistant genes. For future research, it is recommended to explore additional resistance mechanisms and evaluate the effectiveness of new therapeutic strategies. Clinicians should be vigilant about the possibility of clonal dissemination and implement enhanced infection control measures in hospital settings.

Diagnosis of bacteria isolated from patients with cutaneous eczema by PCR technique at Azadi Hospital, Kirkuk, Iraq

Eczema is a common skin condition that can be worsened or caused by a bacterial infection. This study aimed to investigate the bacterial pathogens associated with skin eczema in patients admitted to Azadi Hospital in Kirkuk using polymerase chain reaction (PCR) technology. The goal was to enhance the accuracy and efficiency of bacteria identification compared to traditional methods. A total of [300] patients suffering from symptoms of skin eczema between the ages of 1-40 years, females and males, were included. Bacterial isolates were obtained from skin samples, and conventional microbiological techniques were initially used for identification. Subsequently, molecular analysis using PCR was performed to confirm and characterize the bacterial species. The study included 165 male patients represented (55%), 135 female (45%), of participants who had symptoms of skin eczema. The PCR test targeted specific genes associated with common bacteria implicated in skin infections, such as Staphylococcus. aureus (33 %), Staphylococcus epidermidis (28%), Klebsiella pneumoniae (20 %) , pseudomonas aeruginosa (13 %), respectively, and the remaining( 6 %). of the samples were natural flora. The genetic fingerprints of these bacterial species were identified, an image of them was taken on an agarose gel electrophoreses , and the results were compared with those obtained through traditional methods to evaluate the sensitivity and specificity of PCR in diagnosing bacterial infections in cases of skin eczema. Preliminary results indicated that PCR significantly improved the accuracy and speed of bacterial identification compared to traditional methods. It has allowed the detection of specific bacterial species that might be missed using conventional methods. The prevalence and diversity of bacterial species causing skin eczema in Azadi Hospital in Kirkuk were also explored. This study contributes valuable insights into the microbial etiology of cutaneous eczema, emphasizing the importance of molecular techniques in accurate and rapid diagnosis. The findings may guide more targeted and effective treatment strategies for patients with bacteria-associated skin eczema, ultimately improving patient outcomes and reducing the risk of complications.

Distribution of chaperone-usher fimbriae and curli fimbriae among uropathogenic Escherichia coli

BackgroundIn the present study, we aimed to determine the frequency of the csgA, fimH, mrkD, foc, papaGI, papGII and papGIII genes, to provide and to design fimbrial adhesin gene (FAG) patterns and profiles for the isolated uropathogenic Escherichia coli (UPEC) strains.MethodsThe enrollment of 108 positive urine samples was performed during seven months, between January 2022 and July 2022. The UPEC strains were confirmed through the standard microbiological and biochemical tests. The antimicrobial susceptibility test was performed through the Kirby–Bauer disc diffusion method. Molecular screening of FAGs was done through the polymerase chain reaction technology. The statistical analyses including chi square and Fisher’s exact tests were performed to interpret the obtained results in the present study.ResultsAs the main results, the antimicrobial resistance (AMR) patterns,multi- (MDR) and extensively drug-resistance (XDR) patterns and FAG patterns were designed and provided. fimH (93.3%), csgA (90.4%) and papG (37.5%) (papGII (30.8%)) genes were recognized as the top three FAGs, respectively. Moreover, the frequency of csgA-fimH gene profile was identified as the top FAG pattern (46.2%) among the others. The isolates bearing csgA-fimH gene profile were armed with a versatile of phenotypic AMR patterns. In the current study, 27.8%, 69.4% and 1.9% of the UPEC isolates were detected as extended-spectrum ß-lactamases (ESBLs) producers, MDR and XDR strains, respectively.ConclusionsIn conclusion, detection, providing and designing of patterns and profiles in association with FAGs, AMR feature in UPEC strains give us an effective option to have a successful and influential prevention for both of UTIs initiation and AMR feature.

Biochemical and Molecular Identification of Azospirillum brasilense Bacteria and Evaluation of Their Efficiency in Producing Hormones, Dissolving Phosphorus, and Fixing Nitrogen

The study aimed to isolate A.brasilense bacteria from the soil of the rhizosphere of different plants and different locations in Al-Diwaniyah Governorate. They were identified in two ways. The first was the routine method, which included studying the microscopic and cultural characteristics and biochemical tests of the isolates. The second method was molecular, using polymerase chain reaction (PCR) technology and using primers. It also included testing the efficiency of these isolates in dissolving tricalcium phosphate (TCP) on Pikovskaya agar medium, fixing nitrogen in the liquid nutrient medium (N.B), and measuring the amount of hormone production using an HPLC device. The results of isolation and regular and molecular identification the presence of ten isolates of bacteria bearing the characteristics of A. brasilense bacteria, out of fifteen local bacterial isolates, took the following symbols and sequences (Az2, Az3, Az5, Az6, Az7, Az9, Az11, Az12, Az13, Az14), as the results showed confirmation of the identification of the bacterial isolates identified by biochemical tests. Using a specialized primer to amplify the 462bp fragment of the 16S ribosomal RNA gene, the results of testing the efficiency of the bacteria in dissolving phosphate (TCP) showed that the isolate (Az13) outperformed the highest value in its effectiveness in dissolving metallic phosphorus through the diameter of the clear zone around the colony, which was effective in dissolving phosphate of up to 3.89 mm. As for the nitrogen fixation efficiency test, the isolate (Az3) excelled in the amount of fixed nitrogen reaching 12.44 mg.L-1. As for the amount of its production of growth regulators for Auxins, Gibberellins, and Cytokinins, the isolate (Az3) recorded the highest value in the amount of Auxins production ( Indol-3-acetic acid) its concentration reached 34.4 µg.ml-1, and as for the production of the hormone Gibberellins (GA3), the isolate (Az3) recorded the highest value amounting to 34.7 µg.ml-1, as for the production of the hormone Cytokinins (CK), The isolate (Az11) recorded the highest value, amounting to 28.8 µg.ml-1.

Manual Therapy Improves Fibromyalgia Symptoms by Downregulating SIK1.

Fibromyalgia (FM), classified by ICD-11 with code MG30.0, is a chronic debilitating disease characterized by widespread pain, fatigue, cognitive impairment, sleep, and intestinal alterations, among others. FM affects a large proportion of the worldwide population, with increased prevalence among women. The lack of understanding of its etiology and pathophysiology hampers the development of effective treatments. Our group had developed a manual therapy (MT) pressure-controlled custom manual protocol on FM showing hyperalgesia/allodynia, fatigue, and patient's quality of life benefits in a cohort of 38 FM cases (NCT04174300). With the aim of understanding the therapeutic molecular mechanisms triggered by MT, this study interrogated Peripheral Blood Mononuclear Cell (PBMC) transcriptomes from FM participants in this clinical trial using whole RNA sequencing (RNAseq) and reverse transcription followed by quantitative Polymerase Chain Reaction (RT-qPCR) technologies. The results show that the salt-induced kinase SIK1 gene was consistently downregulated by MT in FM, correlating with improvement of patient symptoms. In addition, this study compared the findings in a non-FM control cohort subjected to the same MT protocol, evidencing that those changes in SIK1 expression with MT only occurred in individuals with FM. This positions SIK1 as a potential biomarker to monitor response to MT and as a therapeutic target of FM, which will be further explored by continuation studies.

Application of microsatellites in population genetic studies of reindeer (Rangifer tarandus) (review)

Over a few past decades, theoretical, analytical, and methodological advances in genetics have revolutionized population genetic research, providing a better understanding of evolutionary processes and the history of populations and species. Methodologically, this progress is largely due to the invention of polymerase chain reaction technology and the introduction of microsatellite DNA markers. This review discusses trends in the use of microsatellite markers as effective tools for solving a wide range of issues in population genetics, conservation and evolutionary biology of the only species of the genus Rangifer – reindeer. Based on the analysis of both experimental and review publications (78 sources) of the scientific teams of the Russian Federation, Canada, the United States of America, Ireland, Japan, China, Norway the first works on the successful amplification of reindeer microsatellites have been summarized. There has been demonstrated the significance of the data of markers for studying intra- and inter-population diversity, differentiation, genetic relationships, the impact of anthropogenic factors on genetic diversity and genetic isolation of populations, as well as for reconstructing the evolutionary history of the various reindeer forms.

The use of free DNA for fetal RHD genotyping in the Rh negative pregnant patient—the time has come

Cell-free DNA (cfDNA) to determine the fetal RHD genotype from the maternal circulation was first described in 1993. High throughput assays using polymerase chain reaction technology were introduced in Europe and gained widespread acceptance in the management of the Rhesus alloimmunized pregnancy. The specificity and sensitivity of these assays approached 99%. As confidence was gained with these results, Scandinavian countries began to employ cfDNA for fetal RHD typing as an integral component of their introduction of antenatal Rhesus immune globulin (RhIG) in non-alloimmunized pregnancies. Since 40% of RhD-negative pregnant women will carry an RhD-negative fetus, doses of RhIG were conserved. Recently two U.S. companies have introduced cfDNA assays for RHD as part of their NIPT assays. Both utilize next generation sequencing and have developed methodologies to detect the aberrant RHD pseudogene and the hybrid RHD-CE-Ds genotype. In addition, excellent correlation studies with either neonatal genotyping or serology have been reported. The manufacturer of RhoGAM® has recently announced a national shortage. . Given the current availability of reliable cfDNA assays for determining the RHD status of the fetus, the time has come to implement this strategy to triage the antenatal use of Rhesus immune globulin in the U.S..

Diagnostic Strategies in the Era of Monkeypox Resurgence: A Comprehensive Analysis.

The resurgence of monkeypox (Mpox), an orthopoxvirus infection closely related to smallpox, presents a significant global health challenge. This study presents a comprehensive overview of Mpox, focusing on its clinical manifestations, diagnostic strategies, and testing methodologies. A thorough review of the literature and available data on Mpox, emphasizing diagnostic assays, clinical indicators, and laboratory testing, constitutes the core of this analysis. The study involves insights from Mpox patients and healthcare professionals engaged in its diagnosis and management. Contextualizing the research within the global spread of Mpox addresses the complexities associated with the diagnosis of the disease. The findings illuminate diverse Mpox diagnostic techniques, encompassing viral culture, immunological methods, serology,quantitative polymerase chain reaction (qPCR), electron microscopy, and advanced technologies such as artificial intelligence (AI) and the GeneXpert system. qPCR is highlighted as the benchmark for MPXV detection and quantification. These diagnostic advancements have significantly enhanced the precision and efficiency of Mpox diagnosis, facilitating prompt identification and treatment of infected individuals. The study underscores the critical importance of accurate and timely diagnosis, proper handling and transportation of clinical specimens, and the imperative for point-of-care (POC) testing to control the global spread of Mpox.

Interventional Effect of Donkey Bone Collagen Peptide Iron Chelate on Cyclophosphamide Induced Immunosuppressive Mice.

Immunodeficiency can disrupt normal physiological activity and function. In this study, donkey bone collagen peptide (DP) and its iron chelate (DPI) were evaluated their potential as immunomodulators in cyclophosphamide (Cytoxan®, CTX)-induced Balb/c mice. The femoral tissue, lymphocytes, and serum from groups of mice were subjected to hematoxylin and eosin (H&E) staining, methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell proliferation assays, and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, a non-targeted metabolomics analysis based on UPLC-MS/MS and a reverse transcription polymerase chain reaction (RT-qPCR) technology were used to explore the specific metabolic pathways of DPI regulating immunocompromise. The results showed that CTX was able to significantly reduce the proliferative activity of mouse splenic lymphocytes and led to abnormal cytokine expression. After DP and DPI interventions, bone marrow tissue damage was significantly improved. In particular, DPI showed the ability to regulate the levels of immune factors more effectively than Fe2+ and DP. Furthermore, metabolomic analysis in both positive and negative ion modes showed that DPI and DP jointly regulated the levels of 20 plasma differential metabolites, while DPI and Fe2+ jointly regulated 14, and all 3 jointly regulated 10. Fe2+ and DP regulated energy metabolism and pyrimidine metabolism pathways, respectively. In contrast, DPI mainly modulated the purine salvage pathway and the JAK/STAT signaling pathway, which are the key to immune function. Therefore, DPI shows more effective immune regulation than Fe2+ and DP alone, and has good application potential in improving immunosuppression.

Fabrication of Cost-Effective Microchip-Based Device Using Sandblasting Technique for Real-Time Multiplex PCR Detection.

The combination of multiplex polymerase chain reaction (mPCR) and microfluidic technologies demonstrates great significance in biomedical applications. However, current microfluidics-based molecular diagnostics face challenges in multi-target detection due to their limited fluorescence channels, complicated fabrication process, and high cost. In this research, we proposed a cost-effective sandblasting method for manufacturing silicon microchips and a chip-based microdevice for field mPCR detection. The atomic force microscopy (AFM) images showed a rough surface of the sandblasted microchips, leading to poor biocompatibility. To relieve the inhibitory effect, we dip-coated a layer of bovine serum albumin (BSA) on the irregular substrate. The optimized coating condition was determined by scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) (65 °C for 60 min). After sufficient coating, we performed on-chip PCR tests with 500 copies/mL Coronavirus Disease 2019 (COVID-19) standard sample within 20 min, and the sandblasted microchip displayed a higher amplification rate compared to dry etching chips. Finally, we achieved a 50 min mPCR for screening five resistance genes of the endophthalmitis pathogens on our microdevices, with strong specificity and reliability. Thus, this sandblasted microchip-based platform not only provides a rapid, accessible, and effective solution for multiplex molecular detection but also enables large-scale microfabrication in a low-cost and convenient way.

Revealing the Viable Microbial Community of Biofilm in a Sewage Treatment System Using Propidium Monoazide Combined with Real-Time PCR and Metagenomics.

Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 102-108 CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA-metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms.

Some Chemical and Biological Methods to Control Pepper Root Rot Disease in Iraq

Background: Pepper growing fields in the Babil and Wasit governorates in Iraq, are suffering from a decline in production due to the spread of root rot diseases. The use of biological control techniques, such as the use of beneficial bacteria in the rhizosphere and natural plant extracts, along with chemical fungicides, has recently contributed to reducing the damage of plant pathogens globally. Methods: In this research, the main cause of root rot disease in pepper plants was isolated and diagnosed using polymerase chain reaction technology using (TEF-1α) gene, antagonistic activities of 75 rhizobacterial isolates, natural extracts of garlic and nerium and the fungicides of Promess and Tecto 500 SC were tested in vitro against the pathogen, then the superior bacterial isolates were identified. The ability of biological and chemical agents to control the disease was tested under greenhouse conditions according to the completely randomized design (CRD). Result: The results of isolation showed that the fungus Fusarium solani was dominance and the isolate Fsw45 was the most virulent. 3 of 75 rhizobacterial isolates outperformed the pathogen in an antagonistic tests and identified as Pseudomonas mendocina, P. fluorescens and Staphylococcus kloosii also the fungicides and botanical extracts showed significant antagonistic activities as well in vitro. Under greenhouse conditions, the fungicide treatments excelled in disease control by 100%. As for the 3 bacterial isolates, they achieved a significant reduction in the percentage of disease incidence and disease severity index, their abilities were higher than the nerium and garlic extracts.

Rapid and sensitive detection of shrimp allergens within 30 min using a new food nucleic acid release agent combined with a fast real-time PCR assay

To achieve fast on-site detection of shrimp allergens in food, this study established a new direct, fast, and real-time quantitative polymerase chain reaction (PCR) technology. The technology completed nucleic acid extraction in 4 min using a new nucleic acid releaser, and the DNA samples were detected using fast qPCR within 25 min (total detection was approximately 30 min). The results indicated that the direct fast qPCR can specifically detect shrimp, with a limit of detection of 0.0001% of shrimp in artificially simulated meat samples and a detection precision variation coefficient (CV value) was less than 5%. It successfully identified shrimp in different types of commercial food samples. As a simple, fast, and sensitive nucleic acid testing technology, it has broad application prospects for the on-site detection of shrimp allergens in food.

A comprehensive review on novel synthetic foods: Potential risk factors, detection strategies, and processing technologies

AbstractNowadays, the food industry is facing challenges due to the simultaneous rise in global warming, population, and food consumption. As the integration of synthetic biology and food science, novel synthetic foods have obtained high attention to address these issues. However, these novel foods may cause potential risks related to human health. Four types of novel synthetic foods, including plant‐based foods, cultured meat, fermented foods, and microalgae‐based foods, were reviewed in the study. The original food sources, consumer acceptance, advantages and disadvantages of these foods were discussed. Furthermore, potential risk factors, such as nutritional, biological, and chemical risk factors, associated with these foods were described and analyzed. Additionally, the current detection methods (e.g., enzyme‐linked immunosorbent assay, biosensors, chromatography, polymerase chain reaction, isothermal amplification, and microfluidic technology) and processing technologies (e.g., microwave treatment, ohmic heating, steam explosion, high hydrostatic pressure, ultrasound, cold plasma, and supercritical carbon dioxide) were reviewed and discussed critically. Nonetheless, it is crucial to continue innovating and developing new detection and processing technologies to effectively evaluate these novel synthetic foods and ensure their safety. Finally, approaches to enhance the quality of these foods were briefly presented. It will provide insights into the development and management of novel synthetic foods for food industry.

Comparison between Molecular and Conventional Methods for the Diagnosis of T. evansi in Humans at Babylon, Iraq

In this study, the presence of Trypanosoma evansi was detected and identified humans in Babylon province in Iraq by blood smears and confirmed by polymerase chain reaction (PCR). Try this study by taking a sample blood from a camel breeders (humans) by Venipuncture from north and south of Babylon. 75 blood samples were collected from humans, adult and young of all sexes. The blood smears result was in humans 0% (0/75). The infection rate using PCR technology was 12% (9/75). This was the first study in Iraq to reveal the presence of the disease. Trypanosoma evansi in camel breeders (humans).

Screening for Antibiotic Resistance Genes in Bacteria and the Presence of Heavy Metals in the Upstream and Downstream Areas of the Wadi Hanifah Valley in Riyadh, Saudi Arabia.

Valley surface water is considered a focal public health concern owing to the presence of multi-drug-resistant bacteria. The distribution of antimicrobial resistance (AMR) bacteria in the surface water is affected by the presence of multiple factors, including antibiotics coming from wastewater discharge or other contaminant sources such as pharmaceuticals, biocides, and heavy metals. Furthermore, there is evidence suggesting that high levels of antibiotic resistance genes (ARGs) can be transferred within bacterial communities under the influence of heavy metal stress. Hence, the primary aim of this study is to investigate the presence of heavy metals and bacterial ARGs in upstream as well as downstream locations of Wadi Hanifah Valley in Riyadh, Saudi Arabia. Sample collection was conducted at eighteen surface water sites within the valley in total. The selection of ARGs was associated with the most common antibiotics, including β-lactam, tetracycline, erythromycin, gentamicin, sulphonamide, chloramphenicol, vancomycin, trimethoprim, and colistin antibiotics, which were detected qualitatively using polymerase chain reaction (PCR) technology. The tested antibiotic resistance genes (ARGs) included (blaNDM-1 (for the antibiotic class Beta-lactamases), mecA (methicillin-resistant Staphylococcus aureus), tet(M) and tet(B) (for the antibiotic class Tetracycline), ampC (for the antibiotic class Beta-lactamases), vanA (for the antibiotic class vancomycin), mcr-1 (for the antibiotic class colistin), erm(B) (for the antibiotic class erythromycin), aac6'-Ie-aph2-Ia (for the antibiotic class Gentamicin), sulII (for the antibiotic class sulphonamide), catII (for the antibiotic class Chlorophincol), and dfrA1 (for the antibiotic class trimethoprim). Moreover, an assessment of the levels of heavy metals such as lithium (Li), beryllium (Be), chromium (Cr), cobalt (Co), arsenic (As), cadmium (Cd), tin (Sn), mercury (Hg), and lead (Pb) was conducted by using inductively coupled plasma mass spectrometry (ICPMS). According to our findings, the concentrations of sulphonamide, erythromycin, and chloramphenicol ARGs (erm(B), sulII, and catII) were observed to be the most elevated. Conversely, two ARGs, namely mecA and mcr-1, were not detected in the samples. Moreover, our data illustrated a significant rise in ARGs in the bacteria of water samples from the upstream sites as compared with the water samples from the downstream sites of Wadi Hanifah Valley. The mean concentration of Li, Be, Cr, Co, As, Cd, Sn, Hg, and Pb in the water samples was estimated to be 37.25 µg/L, 0.02 µg/L, 0.56 µg/L,0.32 µg/L, 0.93 µg/L, 0.01 µg/L, 200.4 µg/L, 0.027 µg/L, and 0.26 µg/L, respectively, for the selected 18 sites. Furthermore, it was revealed that the concentrations of the screened heavy metals in the water samples collected from various sites did not surpass the maximum limits set by the World Health Organization (WHO). In conclusion, this study offers a concise overview of the presence of heavy metals and ARGs in water samples obtained from the Wadi Hanifah Valley in Riyadh, KSA. Such findings will contribute to the ongoing monitoring and future risk assessment of ARGs spread in surface water.

The endometrial cancer detection using non-invasive hypermethylation of CDO1 and CELF4 genes in women with postmenopausal bleeding in Northwest China.

The objective of this study was to verify the clinical predictive performance of methylated cysteine dioxygenase type 1 (CDO1m) and CUGBP Elav-like family member 4 (CELF4m) in endometrial cancer (EC) women with postmenopausal bleeding (PMB). A single-center, prospective, and case-control study was conducted in the Gansu Provincial Maternity and Child-care Hospital with 138 female postmenopausal patients enrolled in 2022. All patients underwent body mass index (BMI) detection, transvaginal ultrasonography (TVUS) detection, carbohydrate antigen 125 detection, and the cervical exfoliated cell CDO1/CELF4 gene methylation detection to analyze the sensitivity, specificity, and accuracy of different screening tests statistically with the biopsy and/or dilation and curettage (D&C) pathological diagnosis under hysteroscopy as the gold standard. There was no significant difference in age between the EC group and the non-EC group, P = 0.492. Using quantitative polymerase chain reaction (qPCR) technology, we validated the CDO1 and CELF4 methylation detection with 87.5% sensitivity and 95.9% specificity as a useful strategy for the triage of women with PMB for the detection of EC. In addition, 100% of type II EC (n = 6) were positively detected by the CDO1 or CELF4 methylation test. The CDO1 and CELF4 methylation test with high specificity as an auxiliary diagnostic tool or alternative method provides physicians with a reference to distinguish between benign and malignant tumors in women with postmenopausal bleeding, to justify the necessity of using invasive methods to confirm diagnosis.