Polymerase Chain Reaction Technique Research Articles

Inhibiting the CB1 receptor in CIH-induced animal model alleviates colon injury

Obstructive sleep apnea (OSA) can lead to intestinal injury, endotoxemia, and disturbance of intestinal flora. Additionally, as a crucial component of the endocannabinoid system, some studies have demonstrated that cannabinoid 1 (CB1) receptors are closely linked to the multiple organ dysfunction triggered by OSA. However, the role of the CB1 receptor in alleviating OSA-induced colon injury remains unclear. Here, through the construction of the OSA classic model, we found that the colon tissue of chronic intermittent hypoxia (CIH)–induced mice exhibited an overexpression of the CB1 receptor. The results of hematoxylin-eosin staining and transmission electron microscopy revealed that inhibition of the CB1 receptor could decrease the gap between the mucosa and muscularis mucosae, alleviate mitochondrial swelling, reduce microvilli shedding, and promote the recovery of tight junctions of CIH-induced mice. Furthermore, CB1 receptor inhibition reduced the levels of metabolic endotoxemia and inflammatory responses, exhibiting significant protective effects on the colon injury caused by CIH. At the molecular level, through western blotting and real-time polymerase chain reaction techniques, we found that inhibiting the CB1 receptor can significantly increase the expression of ZO-1 and Occludin proteins, which are closely related to the maintenance of intestinal mucosal barrier function. Through 16S rRNA high-throughput sequencing and short-chain fatty acid (SCFA) determination, we found that inhibition of the CB1 receptor increased the diversity of the microbial flora and controlled the makeup of intestinal flora. Moreover, butyric acid concentration and the amount of SCFA-producing bacteria, such as Ruminococcaceae and Lachnospiraceae, were both markedly elevated by CB1 receptor inhibition. The results of the spearman correlation study indicated that Lachnospiraceae showed a positive association with both ZO-1 and Occludin but was negatively correlated with the colon CB1 receptor, IL-1β, and TNF-α. According to this study, we found that inhibiting CB1 receptor can improve CIH-induced colon injury by regulating gut microbiota, reducing mucosal damage and promoting tight junction recovery.Key points•CIH leads to overexpression of CB1 receptor in colon tissue.•CIH causes intestinal flora disorder, intestinal mucosal damage, and disruption of tight junctions.•Inhibition of CB1 receptor can alleviate the colon injury caused by CIH through regulating the gut microbiota, reducing mucosal injury, and promoting tight junction recovery.Graphical abstract

Using environmental DNA to elucidate range expansion of bigheaded carp in two Missouri River tributaries in eastern South Dakota

AbstractObjectiveWe used environmental DNA (eDNA) sampling methods to determine the possibility of Bighead Carp Hypophthalmichthys nobilis and Silver Carp H. molitrix (hereafter, bigheaded carp) range expansion above barriers to fish movement in two tributaries of the Missouri River in eastern South Dakota.MethodsWe collected water samples above and below two perceived barriers to fish movement: a natural chain of waterfalls in the Big Sioux River and a spillway at the downstream end of a man‐made reservoir in the Vermillion River. We used filtration methods to collect bigheaded carp eDNA from water samples and implemented real‐time quantitative polymerase chain reaction techniques to quantify the amount of eDNA in each sample. A total of 198 water samples and 35 field blanks were collected over 2 years (2021 and 2022).ResultWe detected bigheaded carp eDNA below both barriers in 2021 and 2022 and above the barrier in the Vermillion River during 2022. The median posterior prediction value for detecting eDNA below both barriers was 75.3% (95% credible interval [CrI] = 73.2–77.2%) for Bighead Carp eDNA and 54.2% (95% CrI = 51.5–56.4%) for Silver Carp eDNA. The probability of detecting eDNA above the barriers was significantly smaller: 1% (95% CrI = 0.01–1.10%) in the Big Sioux River and 1% (95% CrI = 0.01–1.30%) in the Vermillion River.ConclusionThe detection of positive samples above the spillway barrier in the Vermillion River provides the first evidence that bigheaded carp may have expanded their range to habitats upstream of their documented range in eastern South Dakota.

Using PCR and RCA Techniques to Investigate the Variants of Cassava Mosaic Virus and Their Distribution in Ghana

Cassava mosaic disease (CMD) caused by cassava begomoviruses is the major constraint to cassava production in Ghana. The disease is known to cause reduction in root yield. To ascertain the distribution of viruses causing CMD, 95 diseased cassava samples were collected in two agroecological zones of Ghana-Deciduous Forest zone and the Transitional zone. On a scale of 1-5, CMD severity was scored. Mean CMD severity score was 2.9, however there was no significant difference (p > 0.05) between the zones. Averagely, CMD score of > 2.8 in 71% of farms visited was recorded. Polymerase chain reaction (PCR) and rolling circle amplification (RCA) were employed for virus identification. PCR revealed that African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and mixed infections were prevalent in both zones. EACMV-Cameroon strain was also identified to be common within these zones. The transitional zone had the highest percentage of CMD infection. Unamplified samples from PCR were amplified using rolling circle amplification (RCA) technique. Amplification and characterisation of complete genome sequences of two isolates were carried out. The complete genome of 2780 nucleotides from samples showed a high similarity to African cassava mosaic virus-Ghana (ACMV-GH). Sequences clustered with ACMV-Ivory Coast, ACMV Nigeria-Ogo, ACMV-BF, ACMV-UG having > 96% identity. This shows the close relation that exists amongst the ACMV strains in Africa. These findings highlight the need for a continuous survey of CMD to help manage the disease in the country.

Rapid detection of FadA in Fusobacterium nucleatum using the quantitative LAMP colorimetric phenol red method in stool samples from colorectal cancer patients

The study aimed to develop a quantitative colorimetric loop-mediated isothermal amplification technique using the phenol red indicator (QLAMP-PhR) for detecting Fusobacterium nucleatum (Fn) levels in colorectal cancer (CRC) patients and healthy individuals. QLAMP-PhR assays were conducted on 251 stool samples specific for the Fn FadA gene. Six primers were synthesized and utilized with master mix reagents, and a phenol red indicator was employed to enhance the QLAMP-PhR technique. A standard quantitative analysis curve was generated using a logarithmic function (absorbance vs. concentration) by serially diluting the copy number of genomic DNA templates (Fn ATCC25586). The CRC group exhibited a significantly higher abundance of Fn compared to the healthy control group (P < 0.001). These findings suggest that the QLAMP-PhR technique effectively identifies Fn specifically by its gene for the key virulence factor FadA. Additionally, ideas for developing a real-time QLAMP-PhR test were presented. Compared to the traditional polymerase chain reaction (PCR) technique, QLAMP-PhR offers several advantages including rapidity, simplicity, specificity, sensitivity, and cost-effectiveness method that can quantitatively screen for Fn presence in normal populations. The QLAMP-PhR method represents a sensitive and specific amplification assay for the rapid detection of the Fn pathogen. To the best of our knowledge, this study is the first to report the application of QLAMP-PhR for detecting FadA in Fn.

Screening for antifolate and artemisinin resistance in Plasmodium falciparum dried-blood spots from three hospitals of Eritrea

Background Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea’s population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea. Methods Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions. Results Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate. Conclusions The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Therapeutic effect and mechanisms of traditional Chinese medicine compound (Qilong capsule) in the treatment of ischemic stroke

Background Qilong capsule (QLC) is a well-known traditional Chinese medicine compound extensively used in clinical practice. It has been approved by the China's FDA for the treatment of ischemic stroke (IS). In our clinical trial involving QLC (ClinicalTrials.gov identifier: NCT03174535), we observed the potential of QLC to improve neurological function in IS patients at the 24th week, while ensuring their safety. However, the effectiveness of QLC beyond the initial 12-week period remains uncertain, and the precise mechanisms underlying its action in IS have not been fully elucidated.Purpose In order to further explore the clinical efficacy of QLC in treating IS beyond the initial 12-week period and systematically elucidate its underlying mechanisms.Study Design This study employed an interdisciplinary integration strategy that combines post hoc analysis of clinical trials, transcriptome sequencing, integrated bioinformatics analysis, and animal experiments.Methods In this study, we conducted a post-hoc analysis with 2302 participants to evaluate the effectiveness of QLC at the 12th week. The primary outcome was the proportion of patients achieving functional independence at the 12th week, defined as a score of 0–2 on the modified Rankin Scale (mRS), which ranges from 0 (no symptoms) to 6 (death). Subsequently, we employed RNA sequencing (RNA-Seq) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) techniques in the QLC trial to investigate the potential molecular mechanisms underlying the therapeutic effect of QLC in IS. Simultaneously, we utilized integrated bioinformatics analyses driven by external multi-source data and algorithms to further supplement the exploration and validation of QLC's therapeutic mechanism in treating IS. This encompassed network pharmacology analysis and analyses at the mRNA, cellular, and pathway levels focusing on core targets. Additionally, we developed a disease risk prediction model using machine learning. By identifying differentially expressed core genes (DECGs) between the normal and IS groups, we quantitatively predicted IS occurrence. Furthermore, to assess its protective effects and determine the key regulated pathway, we conducted experiments using a middle cerebral artery occlusion and reperfusion (MACO/R) rat model.Results Our findings demonstrated that the combination of QLC and conventional treatment (CT) significantly improved the proportion of patients achieving functional independence (mRS score 0–2) at the 12th week compared to CT alone (n = 2,302, 88.65 % vs 87.33 %, p = 0.3337; n = 600, 91.33 % vs 84.67 %, p = 0.0165). Transcriptome data revealed that the potential underlying mechanism of QLC for IS is related to the regulation of the NF-κB inflammatory pathway. The RT-qPCR results demonstrated that the regulatory trends of key genes, such as MD-2, were consistent with those observed in the RNA-Seq analysis. Integrated bioinformatics analysis elucidated that QLC regulates the NF-κB signaling pathway by identifying core targets, and machine learning was utilized to forecast the risk of IS onset. The MACO/R rat model experiment confirmed that QLC exerts its anti-CIRI effects by inhibiting the MD-2/TLR-4/NF-κB signaling axis.Conclusion: Our interdisciplinary integration study has demonstrated that the combination of QLC with CT exhibits significant superiority over CT alone in improving functional independence in patients at the 12th week. The potential mechanism underlying QLC's therapeutic effect in IS involves the inhibition of the MD-2/TLR4/NF-κB inflammatory signaling pathway, thereby attenuating cerebral ischemia/reperfusion inflammatory injury and facilitating neurofunctional recovery. The novelty and innovative potential of this study primarily lie in the novel finding that QLC significantly enhances the proportion of patients achieving functional independence (mRS score 0–2) at the 12th week. Furthermore, employing a "multilevel-multimethod" integrated research approach, we elucidated the potential mechanism underlying QLC's therapeutic effect in IS.

Advances in human papillomavirusdetection and molecular understanding in head and neck cancers: Implications for clinical management.

Head and neck cancers (HNCs), primarily head and neck squamous cell carcinoma (HNSCC), are associated with high-risk human papillomavirus (HR HPV), notably HPV16 and HPV18. HPV status guides treatment and predicts outcomes, with distinct molecular pathways in HPV-driven HNSCC influencing survival rates. HNC incidence is rising globally, with regional variations reflecting diverse risk factors, including tobacco, alcohol, and HPV infection. Oropharyngeal cancers attributed to HPV have significantly increased, particularly in regions like the United States. The HPV16 genome, characterized by oncoproteins E6 and E7, disrupts crucial cell cycle regulators, including tumor protein p53 (TP53) and retinoblastoma (Rb), contributing to HNSCC pathogenesis. P16 immunohistochemistry (IHC) is a reliable surrogate marker for HPV16 positivity, while in situ hybridizationand polymerase chain reaction (PCR) techniques, notably reverse transcription-quantitative PCR (RT-qPCR), offer sensitive HPV detection. Liquid-based RT-qPCR, especially in saliva, shows promise for noninvasive HPV detection, offering simplicity, cost-effectiveness, and patient compliance. These molecular advancements enhance diagnostic accuracy, guide treatment decisions, and improve patient outcomes in HNC management. In conclusion, advances in HPV detection and molecular understanding have significant clinical management implications. Integrating these advancements into routine practice could ultimately improve patient outcomes.

Silencing of ZNF610 suppresses cell proliferation and migration in lung adenocarcinoma.

Zinc finger proteins (ZNFs) play a significant role in the initiation and progression of tumors. Nevertheless, the specific contribution of ZNF610 to lung adenocarcinoma (LUAD) remains poorly understood. This study sought is to elucidate the role of ZNF610 in LUAD. Transcript data of LUAD were obtained from The Cancer Genome Atlas Program (TCGA) database and processed via R program. The expression of ZNF610 was assessed in various cell lines. To compare the proliferative capacity of cells with or without ZNF610 silencing, CCK8, cell colony formation assay, and Celigo label-free cell counting assay were employed. Furthermore, transwell migration and invasion assays were conducted to evaluate the migratory and invasive abilities of the cells. The expression levels of genes and proteins were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot techniques. In different LUAD cells, the expression level of ZNF610 was found to be significantly higher in LUAD cells compared to MRC-5 and BASE-2B cells. Moreover, the silencing of ZNF610 resulted in a decrease in cell proliferation and migration abilities. Additionally, the apoptosis rate of cells increased upon silencing ZNF610. Notably, the proportion of cells in the G0/G1 phase increased, while the proportion of cells in the S phase decreased following ZNF610 silencing. Finally, β-catenin and snail were identified as downstream targets of ZNF610 in cells. Our findings suggest that silencing ZNF610 could inhibit LUAD cell proliferation and migration, possibly through the downregulation of β-catenin and snail.

Combined use of long-lasting insecticidal nets and Bacillus thuringiensis israelensis larviciding, a promising integrated approach against malaria transmission in northern Côte d'Ivoire

BackgroundThe recent reduction in malaria burden in Côte d’Ivoire is largely attributable to the use of long-lasting insecticidal nets (LLINs). However, this progress is threatened by insecticide resistance and behavioral changes in Anopheles gambiae sensu lato (s.l.) populations and residual malaria transmission, and complementary tools are required. Thus, this study aimed to assess the efficacy of the combined use of LLINs and Bacillus thuringiensis israelensis (Bti), in comparison with LLINs.MethodsThis study was conducted in the health district of Korhogo, northern Côte d'Ivoire, within two study arms (LLIN + Bti arm and LLIN-only arm) from March 2019 to February 2020. In the LLIN + Bti arm, Anopheles larval habitats were treated every fortnight with Bti in addition to the use of LLINs. Mosquito larvae and adults were sampled and identified morphologically to genus and species using standard methods. The members of the An. gambiae complex were determined using a polymerase chain reaction technique. Plasmodium infection in An. gambiae s.l. and malaria incidence in local people was also assessed.ResultsOverall, Anopheles spp. larval density was lower in the LLIN + Bti arm 0.61 [95% CI 0.41–0.81] larva/dip (l/dip) compared with the LLIN-only arm 3.97 [95% CI 3.56–4.38] l/dip (RR = 6.50; 95% CI 5.81–7.29; P < 0.001). The overall biting rate of An. gambiae s.l. was 0.59 [95% CI 0.43–0.75] biting/person/night in the LLIN + Bti arm against 2.97 [95% CI 2.02–3.93] biting/person/night in LLIN-only arm (P < 0.001). Anopheles gambiae s.l. was predominantly identified as An. gambiae sensu stricto (s.s.) (95.1%, n = 293), followed by Anopheles coluzzii (4.9%; n = 15). The human-blood index was 80.5% (n = 389) in study area. EIR was 1.36 infected bites/person/year (ib/p/y) in the LLIN + Bti arm against 47.71 ib/p/y in the LLIN-only arm. Malaria incidence dramatically declined from 291.8‰ (n = 765) to 111.4‰ (n = 292) in LLIN + Bti arm (P < 0.001).ConclusionsThe combined use of LLINs with Bti significantly reduced the incidence of malaria. The LLINs and Bti duo could be a promising integrated approach for effective vector control of An. gambiae for elimination of malaria.

Distribution of SARS-CoV-2 Genomes in Wastewaters and the Associated Potential Infection Risk for Plant Workers in Typical Urban and Peri-Urban Communities of the Buffalo City Region, South Africa.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater has been reported in several studies and similar research can be used as a proxy for an early warning of potential Coronavirus disease 2019 (COVID-19) outbreaks. This study focused on profiling the incidence of SARS-CoV-2 genomes in wastewater samples obtained from facilities located in the Buffalo City Municipality. Raw samples were collected weekly using the grab technique for a period of 48 weeks. Ribonucleic acids were extracted from the samples, using the QIAGEN Powersoil Total RNA Extraction kit, and extracted RNA samples were further profiled for the presence of SARS-CoV-2 genomes using Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) technique. Furthermore, various environmental matrices were utilized to estimate the potential health risk to plant operators associated with exposure to SARS-CoV-2 viral particles using the quantitative microbiological risk assessment (QMRA) model. Our findings revealed the prevalence of SARS-CoV-2 genomes with concentrations that ranged from 0.22 × 103 to 17.60 × 103 genome copies per milliliter (GC/mL). Different exposure scenarios were employed for the QMRA model, and the findings indicate a probability of infection (P(i)) ranging from 0.93% to 37.81% across the study sites. Similarly, the P(i) was highly significant (p < 0.001) for the 20 mL volumetric intake as compared to other volumetric intake scenarios, and high P(i) was also observed in spring, autumn, and winter for all WWTPs. The P(i) was significantly different (p < 0.05) with respect to the different seasons and with respect to different volume scenarios.

Multidisciplinary approach on divergent outcomes in spinal muscular atrophies: comparing DYNC1H1 and SMN1 gene mutations.

Spinal Muscular Atrophy (SMA) emerges as a prominent genetic neuromuscular disorder primarily caused by variants in the survival motor neuron (SMN) gene. However, it is noteworthy that alternative variants impacting DYNC1H1 have also been linked to a subtype known as spinal muscular atrophy lower extremity predominant (SMA-LED). This observation underscores the complexity of SMA and highlights the necessity for tailored, gene-specific management strategies. Our study elucidates how similar approaches to managing SMA can yield distinct outcomes, emphasizing the imperative for personalized gene-based interventions in effectively addressing these conditions.Two patients were referred for further management due to clinical suspicion of type-3 SMA. The definitive diagnosis was confirmed through the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique, as well as whole-exome sequencing (WES).The analysis revealed deletions in exon-7 and 8 of SMN1 in the first patient and a likely pathogenic mutation (NM_001376.5(DYNC1H1):c.1867T > C (NP_001367.2: p.Phe623Leu)) in DYNC1H1 in the second patient. Both patients presented with lower limb muscle weakness. However, while the first patient exhibited a gradual increase in severity over the years, the second patient displayed no progressive symptoms. The management was adjusted accordingly based on the genetic findings.Our observation underscores the complexity of SMA and highlights the necessity for tailored, gene-specific management strategies. Our study elucidates how similar approaches to managing SMA can yield distinct outcomes, emphasizing the imperative for personalized gene-based interventions in effectively addressing these conditions.

Granzyme B Promotes Proliferation, Migration and EMT Process in Gastric Cancer.

Granzyme B (GZMB), a critical member of the Gr gene family, is known to play an essential role in diverse physiological and pathological processes such as inflammation, acute and chronic inflammatory diseases, and cancer progression. In this study, we delve deeper into the role of GZMB within the context of gastric cancer (GC) to examine its expression patterns and functional implications. To accomplish this, we applied a combination of quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry techniques. These methodologies allowed us to accurately gauge GZMB expression levels in GC tissues and investigate their correlation with various clinical-pathological variables. Our secondary focus was to discern the regulatory influence of GZMB on GC cell biology. We used an array of assays including cell counting kit-8 (CCK-8), colony formation, 5-ethynyl-2'-deoxyuridine, and migration assays. The effect of GZMB on gastric cancer progression was further validated through a subcutaneous xenograft mouse model. Our findings underscored that GZMB mRNA and protein levels were upregulated in GC tissues, a feature that showed a significant correlation with GC staging. We also discovered that a decrease in GZMB expression via knockdown experiments suppressed the proliferation and migration capabilities of GC cells. This effect was manifested through diminished expression levels of epithelial-mesenchymal transition (EMT) markers. In stark contrast, the overexpression of GZMB through plasmid transfection appeared to enhance the proliferation and migration abilities of GC cells. This was coupled with an upregulation in EMT expression. Our study concludes by emphasizing that GZMB promotes the growth, migration, and EMT processes in gastric cancer. In vitro, cell-based experiments and in vivo xenograft mouse models confirm this. Our findings provide a more comprehensive understanding of GZMB's role in gastric cancer pathogenesis, potentially opening doors for novel therapeutic strategies targeting this molecular pathway.

ORAI1 Genetic Polymorphism and its Correlation with Erythropoietin Resistance, Hemoglobin, and Erythropoietin Serum Level in Iraqi Patients with Chronic Renal Failure on Hemodialysis

Introduction: Chronic renal failure CRF is a widespread, irreversible disease that develops gradually and is classified into five stages depending on the glomerular filtration rate GFR. Anemia is a considerable complication of CRF that becomes serious as GFR decreases, erythropoietin-stimulating agent ESA is used for treating anemia, but the resistance to it stands against its goal in many patients. Studying the genetic polymorphism in one of the erythropoietin-activated pathway store-operated calcium channels SOCs gene is a trial for investigating the cause of resistance. Method: In this study, 174 subjects were included. Among 112 hemodialysis patients with CRF, 54.5% were non-responders, and the enrolled participants ranged from 20 to 79 years; a single nucleotide polymorphism in the ORAI1 gene was selected. Genotyping was done using an allele-specific polymerase chain reaction technique, and the data was analyzed through the Statistical Package for the Social Sciences. Results: The results showed that hemoglobin levels differ significantly between the genetic groups, while erythropoietin levels showed no significant variation. Conclusion: ORAI1 genetic polymorphism has no significant role in erythropoietin resistance in Iraqi hemodialysis patients with CRF, but this genetic variation significantly affects hemoglobin levels, depending on this study.

Application of direct PCR technique using specified mitochondrial DNAencoded ND4 and D-Loop genes on detecting pork DNA in processed meat products

Adulteration or counterfeiting of food products raises public concerns about the halal issue. Many issues related to food products such as meatballs, sausages, and corned beef use pork for counterfeiting. This study aimed to determine a direct polymerase chain reaction (dPCR) technique for pork DNA detection in processed meat products such as meatballs, sausages, and corned beef in a short time and at a low cost using specific primers, ND4 and D-Loop. The study used a direct PCR technique with variations in the incubation time and temperature of the lysis process on the sample. The concentration of DNA obtained from the lysis results with variations in incubation time and temperature in fresh meat and processed meat products was 66.17-469.23 ng/µL, and the purity of DNA in fresh and processed meat products was 1.70-2.25. ND4 primer was more sensitive to DNA amplification than D-Loop in processed meat products using direct PCR. Meatballs, sausages, and corned beef products written on the packaging using beef did not detect any mixing or addition of pork. Direct PCR can detect pork DNA in processed meat products such as meatballs, sausages and corned beef.

PRECISION AND RELIABILITY OF NANOPLATE DIGITAL PCR SYSTEM FOR PORK DNA IDENTIFICATION AND QUANTIFICATION

The adulteration and mislabeling of food ingredients, particularly meat products, pose a significant concern for consumers. In order to address this issue, it is crucial to use precise and sensitive methods for species identification, mainly when dealing with samples that contain a limited number of copies of DNA. The primary objective of this study is to design a digital nanoplate polymerase chain reaction (PCR) technique for the purpose of quantifying species identification. The primer used in this study was cytochrome b gene. The results showed that target DNA can be amplified using the digital PCR nanoplate until the lowest sample concentration of 0.0013 ng/µL. This study can be applied to enhance species identification processes in food samples by utilizing digital PCR nanoplates.

Development of a TaqMan polymerase chain reaction detection method for the precise identification and quantification of an attenuated Eimeria maxima vaccine strain in poultry.

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve's slope of -3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method's application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method's robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Revealing the pathogenesis of gastric intestinal metaplasia based on the mucosoid air-liquid interface.

Gastric intestinal metaplasia (GIM) is an essential precancerous lesion. Although the reversal of GIM is challenging, it potentially brings a state-to-art strategy for gastric cancer therapeutics (GC). The lack of the appropriate in vitro model limits studies of GIM pathogenesis, which is the issue this work aims to address for further studies. The air-liquid interface (ALI) model was adopted for the long-term culture of GIM cells in the present work. This study conducted Immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR), transcriptomic sequencing, and mucoproteomic sequencing (MS) techniques to identify the pathways for differential expressed genes (DEGs) enrichment among different groups, furthermore, to verify novel biomarkers of GIM cells. Our study suggests that GIM-ALI model is analog to the innate GIM cells, which thus can be used for mucus collection and drug screening. We found genes MUC17, CDA, TRIM15, TBX3, FLVCR2, ONECUT2, ACY3, NMUR2, and MAL2 were highly expressed in GIM cells, while GLDN, SLC5A5, MAL, and MALAT1 showed down-regulated, which can be used as potential biomarkers for GIM cells. In parallel, these genes that highly expressed in GIM samples were mainly involved in cancer-related pathways, such as the MAPK signal pathway and oxidative phosphorylation signal pathway. The ALI model is validated for the first time for the in vitro study of GIM. GIM-ALI model is a novel in vitro model that can mimic the tissue micro-environment in GIM patients and further provide an avenue for studying the characteristics of GIM mucus. Our study identified new markers of GIM as well as pathways associated with GIM, which provides outstanding insight for exploring GIM pathogenesis and potentially other related conditions.

Seroepidemiological Investigation of Hepatitis B and C Prevalence and Associated Factors Among People in Custody at Zahedan Central Prison.

On a global scale, approximately 350 million are affected by hepatitis B, and 71 million by hepatitis C. People in custody face elevated risks for these infections. The prevalence and risk factors in Iranian prisons are insufficiently documented. The principal objective of this study was to ascertain the prevalence of hepatitis B and C, coupled with the identification of pertinent influencing factors, within the confines of Zahedan central prison, situated in the southeastern region of Iran. In 2019, we conducted an analytical cross-sectional study involving 407 people in custody, using stratified random sampling. To definitively diagnose hepatitis C virus (HCV) infection (P<0.05), a checklist developed by the researchers, along with enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) techniques, were employed. This study comprised 406 participants (96.3% male) with a median age of 32 years (27-38). Approximately 62% were married, and a substantial proportion of the participants had low education levels (47%), unemployment (64%), and belonged to the Baloch ethnicity (64%). The overall prevalence of hepatitis C and B infections was 2.7% and 10.6%, respectively. Tattooing (adjusted odds ratio [AOR]: 2.07, 95% CI: 1.9-4.5) and marriage (AOR: 1.78, 95% CI: 1.05-3.04) were identified as risk factors for hepatitis B. Moreover, hepatitis C showed a statistically significant association with a family history of hepatitis B and C (AOR: 3.31, 95% CI: 3.93-24.64) and intravenous (IV) drug use (AOR: 7.01, 95% CI: 1.52-32.78) according to the multivariable logistic regression analysis. The prevalence of hepatitis B and C was higher among people in custody in Zahedan central prison. Consequently, targeted interventions are vital to address and reduce viral hepatitis burden in custodial settings.

Assessment of Biofilm Production and Antibiotic Sensitivity Patterns in Klebsiella pneumoniae Isolates from Al-Qadisiyah Hospitals, Iraq

Background: Klebsiella pneumoniae is one of the most common causative agents of nosocomial infections. Opportunistic pathogens can generate a thick layer of biofilm as an important virulence factor. Objectives: The current study was aimed in the detection of biofilm formation in Klebsiella pneumoniae pathogenic capability as a common opportunistic pathogen accounting pneumonia, urinary tract infections, with in nosocomial infections. Materials and Methods: In this observational study, a total of 140 clinical samples obtained from patients with bacterial infections were analyzed. The identification of Klebsiella pneumoniae isolates was performed using selective culture media and biochemical tests. Additionally, biofilm strains were characterized using the Crystal Violet assay and polymerase chain reaction (PCR) techniques. Results: Among the 140 samples collected from various specimens, a total of 100 isolates (43.47%) were identified as Klebsiella pneumoniae culturing and biochemical tests. Out of these isolates, 58 (58%) were obtained from male individuals, while 42 (42%) were obtained from female individuals. Using the phenotypic method, the analysis revealed that 18% isolates were classified as strong biofilm producers, 33% as medium biofilm producers, 49% as weak biofilm producers, and 30 as non-biofilm producers. The frequency of specific genes in the isolates was reported as follows: wzm (47%) and markA (69%). Conclusion: The presence of the markA gene is significant in the context of biofilm formation in Klebsiella pneumoniae strains, as it serves as a marker for distinguishing various types of biofilms.

Association of Polymorphism with Periodontitis and Salivary Levels of Hypoxia-Inducible Factor-1α.

This investigation aims to investigate the association between HIF-1α genetic polymorphism and periodontitis and examine and contrast the levels of HIF-1α present in the saliva of subjects afflicted with periodontitis and in the control group. Additionally, this study aims to establish diagnostic proficiency of this biomarker in distinguishing between periodontal health and disease. This study entailed the collection of venous blood samples and unstimulated saliva samples from a total of 160 participants, encompassing 80 individuals diagnosed with periodontitis and 80 periodontitis-free individuals. The periodontal parameters were evaluated, involving the measurement of clinical attachment loss, the probing pocket depth, and the bleeding on probing percentage. Subsequently, genetic analysis of HIF-1α using polymerase chain reaction (PCR) technique, DNA sequencing, and enzyme-linked immunosorbent assays was conducted. The genetic analysis of 352 bp of the HIF-1α gene revealed the presence of 66 single-nucleotide polymorphisms (SNPs) in control samples, whereas 78 SNPs were found in periodontitis sample. The nucleotide A was replaced with a C nucleotide at position 207 of the amplified PCR fragments. The homozygous AA pattern was predominant in the control group, with significant differences between the two groups. In contrast, the homozygous CC pattern was more dominant in the periodontitis group, with significant differences between the two groups. The analysis of Hardy-Weinberg equilibrium for the comparison between the observed and the expected genotypes showed significant differences between the observed and the expected values in the control and periodontitis groups, as well as the total sample. The highest mean values of the measured periodontal parameters were found in the periodontitis group (clinical attachment loss = 4.759, probing pocket depth = 4.050, and bleeding on probing = 30.950) with statistically significant differences between the groups. The periodontitis group showed significantly higher salivary HIF-1α levels compared to control group (p < 0.001). Besides, HIF-1α is a good biomarker in distinguishing between periodontal health and periodontitis. rs1951795 SNP of HIF-1α has no significant impact on the progression of periodontitis and the salivary level HIF-1α. Periodontitis results in a notable elevation in HIF-1α salivary levels, with an outstanding diagnostic ability to distinguish between periodontitis and periodontal health.