Polymerase Chain Reaction Technology Research Articles

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.

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.

Systematic assessment of the reliability of quantitative PCR assays targeting IS900 for the detection of Mycobacterium avium ssp. paratuberculosis presence in animal and environmental samples.

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's Disease (JD), which is endemic to dairy cattle and also incriminated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. JD is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative polymerase chain reaction (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD disease. MAP IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry and TaqMan technology was used in 107 reports. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.

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.

Investigating the Presence of Rotavirus in Wastewater Samples of Bhopal Region, India, by Utilizing Droplet Digital Polymerase Chain Reaction.

Rotavirus-induced viral gastroenteritis outbreaks result in over two million hospitalizations globally yearly. Wastewater-based epidemiology (WBE) has emerged as a crucial tool for detecting and monitoring viral outbreaks. The adoption of WBE has been instrumental in the early detection and surveillance of such viral outbreaks, providing a non-invasive method to assess public health. This study aims to utilize droplet digital polymerase chain reaction (ddPCR) technology to detect and quantify Rotavirus in wastewater samples collected from the Bhopal region of India, thereby contributing to the understanding and management of viral gastroenteritis outbreaks through environmental surveillance. In this study, we used ddPCR to detect and quantify Rotavirus in wastewater samples collected from the Bhopal region of India. We monitored its viral presence in municipal sewage treatment plants bi-weekly using an advanced ddPCR assay. Targeting the rotavirus non-structural protein 3 (NSP-3) region with custom primers and TaqMan probes, we detected virus concentration employing polyethylene glycol (PEG). Following RNA isolation, complementary DNA (cDNA) synthesis, and ddPCR analysis, our novel method eliminated standard curve dependence, propelling virus research and treatment forward. Out of the 42 samples collected, a 16.60% positivity rate was observed, indicating a moderate presence of Rotavirus in Bhopal. The wastewater treatment plants (WWTP) attached to a hospital exhibited a 42.85% positivity rate, indicating the need for targeted monitoring. Leveraging ddPCR, precise quantification of rotavirus concentrations (ranging from 0.75 to 28.9 copies/µL) facilitated understanding and supported effective remediation. This study emphasizes the importance of vigilant wastewater surveillance, especially in WWTPs with higher rotavirus prevalence. The significance of ddPCR in comparison to conventional and real-time PCR lies in its superior sensitivity and specificity in detecting and quantifying positive samples. Furthermore, it can identify positive samples even in the smallest quantities without the need for a standard curve to evaluate. This makes ddPCR a valuable tool for accurate and precise detection and quantification of samples.

Association of gene polymorphisms and the decreased expression of long non-coding RNA LOC553103 with rheumatoid arthritis.

Long non-coding RNAs (lncRNAs) are involved in many key bioprocesses, including the occurrence and development of rheumatoid arthritis (RA). We aimed to analyze the association of genetic variants of long non-coding RNA LOC553103 and its peripheral blood mononuclear cells (PBMC) expression with RA. We enrolled 457 RA patients and 551 healthy controls and conducted a case-control study to analyze the relationship between LOC553103 gene rs272879 and the susceptibility of RA by TaqMan single nucleotide polymorphism genotyping. Among them, we sampled 92 cases and 92 controls, respectively, to detect the PBMC level of LOC553103 using quantitative real-time polymerase chain reaction technology. We explored the association between LOC553103 rs272879 and its PBMC expression levels in 71 RA patients. Mann-Whitney, Chi-square, and Spearman correlation analysis were used for statistical analysis and P-value <.05 was considered statistically significant. The genotype frequency of LOC553103 rs272879 CC was increased, and CG was decreased in RA patients compared to the control group (χ2 = 6.772, P = .034). The LOC553103 expression level in PBMC of RA patients was downregulated compared to healthy control (Z = -4.497, P < .001). Moreover, negative correlations were observed between the PBMC level of LOC553103 and erythrocyte sedimentation rate (rs = -0.262, P = .018), white blood cell count (rs = -0.382, P = .004), platelet (rs = -0.293, P = .030), and disease activity score in 28 joints (rs = -0.271, P = .016) in RA patients. This study provides the first evidence supporting an association between LOC553103 gene polymorphisms and susceptibility of RA and a relationship of PBMC level of LOC553103 with clinical manifestations and laboratory indicators of RA patients.

Development of methods to analyse Plasmodium falciparum single nucleotide polymorphisms in PfCRT (А &gt; C), PfMDR1 (A &gt; T) and PfDHFR (G &gt; A) genes that determine resistance to quinoline, diamino-pyrimidine and sulfonamide groups of antimalarial drugs

BACKGROUND: The drug resistance of tropical malaria pathogens to mefloquine, chloroquine, pyrimethamine and their derivatives is associated with three single nucleotide polymorphisms: K76T (A403627C), S1034C (A960989T) and S108N (G748410A). These mutations are linked to changes in the structure of the PfCRT and PfMDR1 genes of Plasmodium falciparum.&#x0D; AIM: Develop methods for identifying single nucleotide polymorphisms that are suitable for early diagnosis of drug-resistant forms of tropical malaria.&#x0D; RESULTS: A method was developed based on restriction fragment length analysis using ApoI endonuclease to detect the K76T polymorphism (A403627C). The criterion for determining parasite resistance to chloroquine was the appearance of a single 145 bp band on the electropherogram. The genotype of the pathogens remained unchanged and their drug sensitivity was preserved, as indicated by the separation of two fragments of 98 and 47 bp.&#x0D; A system for detecting S108N (G748410A) was developed using the Bse1I endonuclease. The appearance of a single 507 bp band on the electropherogram indicated the mutant genotype of the pathogens, while the appearance of two fragments (323 and 184 bp) indicated an unchanged genotype and preservation of drug sensitivity of plasmodiae.&#x0D; To identify the A T polymorphism in the PfMDR1 gene at position 960989, polymerase chain reaction technology will be used with two allele-specific primers. One primer will detect the wild-type allele, and the other will detect the mutant genotype. The amplifiable fragment of the PfMDR1 gene contains sequences of the 1034th codon. Depending on the P. falciparum genotype, 261 bp fragments will be obtained with one of the allele-specific primers.&#x0D; CONCLUSION: Criteria for assessing drug resistance of P. falciparum were developed based on the analysis of obtained data. Haplotypes K76T (band 145 bp) and S1034C (band 262 bp with the direct primer S1034C-F2) serve as indicators of the relative resistance of pathogens to chloroquine, mefloquine, and their derivatives. Positive results of examination for haplotype S108N (bands 323 and 184 bp) should be considered as a sign of decreased sensitivity to pyrimethamine. The developed methods can be used in clinical practice and for epidemiological monitoring.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81kcal/mol, while the docking energy for meropenem was - 4.92kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28kcal/mol, compared with - 4.60kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16µg/mL, whereas levofloxacin-resistant strains had MIC values of 8µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2h under the action of 8µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

The Use of Next Generation Sequencing to Detect Early Implant Infections: The Future of Microbiology and Targeted Antibiotic Treatment

Abstract Goals/Purpose The presence of fluid around foreign bodies, particularly breast implants, is a well-established phenomenon; However scant data exists regarding the composition of this fluid. The difficulty in studying this fluid arises from the lack of safe access to aspirate fluid post-breast augmentation, and by the loss of sterility once fluid exits the body via a drain bulb as traditionally placed during breast reconstruction. Our innovative breast reconstruction approach utilizes a drainless technique with a dual chamber tissue expander (TE) in the pre-pectoral plane, offering a unique avenue to sterilely aspirate peri-implant fluid through a percutaneous drainage port. In this study, we employ Next Generation Sequencing (NGS), utilizing polymerase chain reaction technology, to discern the composition of microbial DNA within peri-prosthetic breast implant fluid. NGS, known for its precision and cost-effectiveness, circumvents the biases inherent in traditional culture methods. By integrating this cutting-edge technology with our breast reconstruction methodology, we sought to study peri-prosthetic breast implant fluid to better understand when implant infections arise and if peri-prosthetic fluid could detect infections before clinical symptoms arise. Methods/Technique This is a single surgeon, prospective study of patients undergoing mastectomy, followed by pre-pectoral, drainless, TE reconstruction. Peri-prosthetic fluid was collected through the TE drainage port in the OR immediately after closure, at 1 week post op, and 3 weeks post op. This fluid was sent for traditional culture and NGS. Patients were observed for signs of infection, antibiotics, return to OR, or implant loss. Results/Complications 33 breasts from 20 patients were included. Patients had a mean age of 50 years. The average length of drainage was 21 days post-operative, with a mean of 131 cc of fluid drained per breast per week. Eight breasts had concern for skin necrosis. In 25 breasts (76%), complete sterility was achieved at the end of the case with no NGS detection of microorganisms in the initial peri-implant fluid sample. However, NGS detected microorganisms in the initial sample in 8 breasts (24%). In 5 of these 8 breasts, microorganisms were cleared by the 1-week sample and another 2 cleared by 3 weeks. The remaining 1 breast continued to have positive NGS and developed clinical signs of infections by week 2, which were not picked up by traditional culture until week 3. One breast with skin necrosis developed a positive NGS at week 3 and clinical signs of infection at week 4. No other clinical infections were seen throughout the study. Conclusion We are one of the first groups to demonstrate the ability to achieve peri-implant sterility at the time of implant insertion in the operating room. This could have significant implications for understanding the necessity of peri-operative antibiotics following breast implant placement. Our investigation into the microbial composition of ostensibly "sterile" peri-prosthetic breast implant fluid provides insight into when microorganisms arise and when they become clinical infections. These promising preliminary results suggests that peri-prosthetic fluid monitoring through Next-Generation Sequencing (NGS) holds significant potential in preemptively detecting implant infections before the onset of clinical symptoms, thereby reducing infection rates. Furthermore, our findings open avenues for targeted antibiotic therapy, contributing to the imperative goal of antibiotic stewardship. Ongoing work is beginning to explore the impact of microorganisms within the fluid on breast capsule formation, as we strive to enhance our understanding of breast implants and infections.

Pasteurized Akkermansia muciniphila and its outer membrane protein Amuc_1100 alleviate alcoholic liver disease through modulating gut microbiota and host metabolism

Recent researches have reported that pasteurized Akkermansia muciniphila, as a paraprobiotic, still plays an active role in various metabolic diseases. However, its role in alcoholic liver disease (ALD) remains unexplored. This study aims to evaluate the impact and mechanisms of pasteurized A. muciniphila on ALD. Its effects on ALD were assessed by phenotypic, biochemical parameters, and histological features in a mouse model. Further investigations into underlying mechanisms were conducted through gut microbiome, metabolomics analyses and quantitative real-time polymerase chain reaction (qRT-PCR) technology. Heterologously expressed outer membrane protein Amuc_1100 was utilized to explore the molecular action mechanism. Our research demonstrated that pasteurized A. muciniphila can indeed alleviate alcohol-induced liver damage by modulation of gut microbiota and host metabolism. It suppressed the proliferation of pathogenic microbes and fostered the growth of probiotics. Additionally, it rectified alcohol-induced abnormalities in serum metabolic patterns, particularly in lipid metabolism. Specifically, pasteurized A. muciniphila elevated serum levels of 3-hydroxybutyric acid, palmitoleic acid, acetylcarnitine, DHA, and arachidonic acid in alcohol-fed mice. qRT-PCR results showed that it reversed the alcohol-induced upregulation of mRNA expression of hepatic fatty acid synthesis genes and decreased mRNA expression of fatty acid oxidation and transport genes. Importantly, we identified that Amuc_1100 could partially recapitulate the protective effects of pasteurized A. muciniphila on ALD, especially regarding hepatic lipid metabolism. This study enhances our comprehension of the function of pasteurized A. muciniphila and underscores its potential therapeutic application, along with Amuc_1100, in the treatment of ALD.

Correlation analysis of serum miR-145 and miR-210 with carotid artery stenosis and their predictive value for cerebral ischemic events

Objective To analyze the significance of serum miR-145 and miR-210 expression levels in the diagnosis of carotid artery stenosis. Methods During the same period, 55 healthy individuals who received physical examination in the same hospital were recruited as controls and assigned to a non-stenosis group. Among the included patients, there were 45 cases of mild stenosis, 14 cases of moderate stenosis, and 6 cases of severe stenosis after carotid color Doppler ultrasonography. The expression levels of miR-145 and miR-210 in serum were measured using real-time fluorescence quantitative polymerase chain reaction (qPCR) technology. Results The expression levels of serum miR-145 and miR-210 in carotid artery stenosis group were significantly lower than those in non-stenosis group (p < 0.001). Multivariate Logistic regression analysis showed that smoking history, diabetes, hypertension and total cholesterol were positively correlated with the occurrence of carotid artery stenosis (p < 0.05). The expression levels of miR-145 and miR-210 were significantly negatively correlated with carotid artery stenosis (p < 0.001). In addition, patients with carotid artery stenosis and low expression levels of miR-145 and miR-210 had a greater risk of cerebral ischemia (p < 0.05). Cox regression analysis showed that the low expression of miR-145 and miR-210 were independent predictors of cerebral ischemic events. ROC analysis confirmed that miR-145 and miR-210 had good diagnostic efficacy in cerebral ischemia (p < 0.001). Conclusion The decreased expression of miR-145 and miR-210 in serum is closely related to the diagnostic significance of carotid artery stenosis, and may be used to predict the occurrence of cerebral ischemic events.

A cost-effective and field-deployable sensing system for chip-integrated detection of bacteria with the naked eye

To tailor the broadly applied polymerase chain reaction (PCR) technology to point-of-care settings, an intuitive, time-saving, and cost-effective method to determine the DNA amplification results has significant practical potential. Herein we present an economical and field-deployable sensing system that enables naked-eye readout of PCR results. The proposed system is powered by an amplification enhancer to greatly enhance the positive-to-background ratio and a palm-sized readout device to assist on-site applications. High specificity, as well as single-copy sensitivity, was achieved in detecting Salmonella enterica and other bacterial pathogens. Using the proposed system, we identified the pathogenic bacterial strains in artificially contaminated food samples easily, quickly, and reliably. Also crucial for point-of-care testing, this naked-eye readout system applies to microfluidic platforms with the sample volumes scaled down to 1 μL. The time savings, portability, and cost-effectiveness of the established sensing system make it transformative for on-chip bacteria detection in point-of-care and field settings, especially in resource-limited areas.

It was not possible to detect BRAF V600E mutation in circulating cell-free DNA from patients with ameloblastoma: A diagnostic accuracy study.

The objective of this study is to evaluate the diagnostic accuracy of plasma-based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell-free DNA from patients with ameloblastoma. This is a prospective diagnostic accuracy study conducted based on the Standards for Reporting Diagnostic Accuracy recommendations. The index test was the plasma-based liquid biopsy, whereas the reference standard was the conventional tissue biopsy. The target condition was the detection of BRAF V600E mutation. The study population consisted of individuals with ameloblastoma recruited from three tertiary hospitals from Brazil. A negative control group composed of three individuals with confirmed wild-type BRAF lesions were included. The participants underwent plasma circulating cell-free DNA and tumor tissue DNA isolation, and both were submitted to using competitive allele-specific TaqMan™ real-time polymerase chain reaction technology mutation detection assays. Sensitivity and specificity measures and positive and negative predictive values were calculated. Twelve patients with conventional ameloblastoma were included. BRAF V600E mutation was detected in 11/12 (91.66%) ameloblastoma tissue samples. However, the mutation was not detected in any of the plasma-based liquid biopsy circulating cell-free DNA samples in both ameloblastomas and negative control group. The sensitivity and specificity of plasma-based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell-free DNA was 0.0 and 1.0, respectively. The agreement between index test and reference standard results was 26.66%. Plasma-based liquid biopsy does not seem to be an accurate method for the detection of the BRAF V600E mutation in circulating circulating cell-free DNA from patients with ameloblastoma, regardless of tumor size, anatomic location, recurrence status, and other clinicopathological features.

USE THE NANO-CHITOSAN TO INHIBIT THE TWO FUNGI ASPERGILLUS NIGER. AND NIGROSPORA SPHAERICA WHICH ISOLATED FROM ZEA MAYS L. LABORATORY

The isolation from Zea mays L. showed two types of fungi Aspergillus niger., Nigrospora sphaerica with a frequency of 38% and 22 %sequentially, Fungi were Isolated diagnosed by keys of taxonomic, Diagnosis of the fungus was assured N. sphaerica depending on the Polymerase Chain Reaction Technology {PCR}. It is the first recording in Iraq/ Karbala Province. Nano-chitosan achieved 100% inhibition rates in vitro

Polymerase Chain Reaction Microchip and PID Controller Based Thermal Cycler Design

Numerous specific procedures are needed for nucleic acid detection using traditional approaches. Because of the time and instrumentation requirements, poor detection limits, and other factors, they are behind in creating point-of-care (POC) testing. In addition, some of these traditional approaches can be extremely sensitive. Microfluidic technologies are being used to overcome these challenges by enabling the efficient and automatic performance of all analytical stages, such as sample pre-treatment, reactions, separations, and diagnostics in microchannels on a small chip. The use of microfluidics technology in polymerase chain reaction (PCR) technology has resulted in many advantages. PCR microchips and portable thermal cyclers that can be used for POC testing have enabled faster amplification with accurate results. This paper proposes a low-cost, fast enough thermal cycler for POC testing that uses a polymer-glass microchip and an affordable Peltier element to quickly heat samples. A laser cutter was used to build the microchip needed to perform PCR, which was designed using a computer-aided design (CAD) software. The heating and cooling elements employed in the creation of the thermal cycler were Peltier elements, heat sink, centrifugal fan, and CPU fan. A microcontroller and H-Bridge modules were used to control the temperature with the help of proportional-integral-derivative (PID) controller algorithm. After that, the circuit components and PID control algorithm—which was created for temperature control—were combined to create the PCR cycle. The analysis focuses on the heating and cooling performances of suggested thermal cycler.

In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou.

Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 μg/mL, and minimum bactericidal concentration (MBC) values were 160-320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.