Polymerase Chain Reaction Amplification Research Articles

Morphological and Molecular Characterization of Trichotylenchus dispersus (Nematoda: Dolichodoridae), a Newly Recorded Plant-Parasitic Nematode in the Rhizosphere Soil of Tomato Plants in Thailand

Trichotylenchus sp. is a migratory ectoparasitic nematode (PPN) that causes hypoplasia disease symptoms in economic plants, such as maize and banana. In this study, soil samples were collected from 6 locations in a tomato field in Khon Kaen Province, Thailand, for the purpose of nematode extraction using the Cobb’s sieving and flotation-centrifugation techniques. Morphological and molecular characterization of the collected PPN identified it as Trichotylenchus sp., a PPN not previously found in Thai tomato fields. Morphologically, the nematode has a C-shaped body, a rounded lip region, robust stylet and dorsal gland orifice located 2.0 ± 0.5 µm from the base of basal knobs. The tail is conically shaped, and the cuticle exhibits annulations with 3 incisures in the lateral field. The morphological characteristics observed closely matched Trichotylenchus sp. To confirm the identity, diagnosis was done using Polymerase Chain Reaction (PCR) molecular technique. Nematode DNA amplification was conducted with different target genes using primer sets AB28/TW81 for ITS1-5.8S-ITS2 and D2A/D3B for D2-D3 region of the 28S rRNA. The PCR amplification showed DNA fragments of approximately 950 and 800 bp, respectively. The sequences obtained were compared with those in the NCBI GenBank, which showed a 98.0 - 99.4 % identity match with Trichotylenchus dispersus specimens previously documented in China. In addition, the phylogenetic trees reiterated a nematode grouping with T. dispersus, 100 % bootstrap value. To the best of our knowledge, this is the first report of the presence of T. dispersus in the soils of tomato field in Thailand. This nematode is likely to become a major factor limiting tomato production yields if not properly managed. HIGHLIGHTS Six plant-parasitic nematodes (PPNs) were found to be associated with the tomato gown in field. A newly recorded PPN in the genus, Trichotylenchus, found in Thailand, has been documented in this study. Morphological and molecular characterization identified PPN as Trichotylenchus disperses. This is the first report on incidence of dispersus in a tomato field in Thailand. GRAPHICAL ABSTRACT

Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips.

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γLV) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R2 values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.

Streamlined boiling lysis DNA extraction for Gram-positive aquaculture pathogen Streptococcus agalactiae.

Accurate genetic analysis is essential for the detection of pathogens as it necessitates suitable DNA extraction methods tailored to specific microorganisms such as Gram-positive bacteria. This study examined several commercial and simplified DNA extraction methods for their suitability in isothermal downstream applications. Extracted DNA was assessed using spectrophotometry, electrophoresis, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) while its stability was inspected after five months of storage. The findings revealed variations in DNA yield, purity and integrity among the extraction methods. While extraction kits demonstrated high yield and purity, the in-house extraction techniques showed incoherent correlation between yield and purity, yet showed promise for a streamlined extraction process. The DNA acquired from all methods yielded positive amplification in PCR and LAMP. DNA extracted by kits exhibits prolonged stability than those obtained via boiling lysis. Both methods offer distinct advantages, with commercial kits providing longer stability and high-quality DNA while boiling lysis stands out for its simplicity, with shorter handling and processing periods. This study emphasizes selecting ideal extraction methods for Streptococcus agalactiae, in the prospect of aquaculture settings. In particular, successful LAMP reaction suggests that boiled extracts are feasible enough for detection, and suited for point-of-care (POC) testing where prompt detection of aquatic pathogens is often critical. Ultimately, the choice of method should be contemplated on a case-by-case basis such as the study goals, intended settings, and type of samples.

Repeat-mediated recombination results in Complex DNA structure of the mitochondrial genome of Trachelospermum jasminoides

BackgroundTrachelospermum jasminoides has medicinal and ornamental value and is widely distributed in China. Although the chloroplast genome has been documented, the mitochondrial genome has not yet been studied.ResultsThe mitochondrial genome of T. jasminoides was assembled and functionally annotated using Illumina and nanopore reads. The mitochondrial genome comprises a master circular molecular structure of 605,764 bp and encodes 65 genes: 39 protein-coding genes, 23 transfer RNA (tRNA) genes and 3 ribosomal RNA genes. In addition to the single circular conformation, we found many alternative conformations of the T. jasminoides mitochondrial genome mediated by 42 repetitive sequences. Six repetitive sequences (DRS01–DRS06) were supported by nanopore long reads, polymerase chain reaction (PCR) amplifications, and Sanger sequencing of the PCR products. Eleven homologous fragments were identified by comparing the mitochondrial and chloroplast genome sequences, including three complete tRNA genes. Moreover, 531 edited RNA sites were identified in the protein-coding sequences based on RNA sequencing data, with nad4 having the highest number of sites (54).ConclusionTo our knowledge, this is the first description of the mitochondrial genome of T. jasminoides. Our results demonstrate the existence of multiple conformations. These findings lay a foundation for understanding the genetics and evolutionary dynamics of Apocynaceae.

Streamlining RNA Aptamer Selection via Unique Molecular Identifiers and High-Throughput Sequencing.

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.

Isolation and pathogenicity of a fowl adenovirus 8b (FAdV-8b) strain in Cherry Valley ducks

ABSTRACT Inclusion body hepatitis (IBH) is an economically important viral disease primarily affecting the poultry industry. In this study, we isolated a strain of FAdV-8b (strain SDYT) from naturally infected ducks and the hexon and fiber gene sequences were analysed by polymerase chain reaction (PCR) amplification. In order to study the pathogenicity of FAdV-8b in Cherry Valley ducks, we inoculated 10- and 20-day-old ducks with 0.3 ml of FAdV-4 virus (TCID50 of 105.5/0.1 ml) either orally or intramuscularly. Clinical signs, gross lesions and histopathological changes, cytokines, viral load and antibody levels were observed and recorded within 15 days after infection. Pathomorphological investigations revealed that ducks in the experimental group exhibited hepatitis. Histopathology showed multiple organ damage, including serious liver and kidney lesions. Furthermore, elevated levels of inflammatory cytokines and antibodies were noticed, due to the infection and innate immune response. At a later stage of infection, immunosuppression occurred, resulting in decreased levels of cytokines. Determination of viral load showed that the virus was present in several organs, with the highest viral DNA load found in the liver, followed by the kidney. Compared to birds infected orally, the intramuscular group exhibited the highest viral load. In summary, this study increases our understanding of the pathogenicity of FAdV-8b in ducks and establishes a model that will inform antiviral drug testing and vaccine evaluation for IBH, thereby preventing and reducing the spread of IBH in the poultry industry. RESEARCH HIGHLIGHTS A strain (SDYT) of fowl adenovirus 8b (FAdV-8b) was successfully isolated from ducks. Cherry Valley ducks were successfully infected with FAdV-8b. Different routes of infection can lead to duck mortality, more pronounced when birds are injected intramuscularly. FAdV-8b (SDYT) was distributed in various tissues and organs of ducks, causing different degrees of lesions.

Clinical significance of Spinal Muscular Atrophy carrier detection in Guangdong Province, China: Insights from quantitative polymerase chain reaction and multiplex ligation-dependent probe amplification analysis

ObjectiveThis study aimed to identify carriers of Spinal Muscular Atrophy (SMA) among 10,630 pregnant women in Guangdong Province and provide prenatal diagnoses for high-risk fetuses from carrier couples. The goal was to prevent the birth of children affected by SMA. We evaluated the effectiveness of quantitative PCR (qPCR) and multiplex ligation-dependent probe amplification (MLPA) in detecting deletions in the SMN1 gene, with MLPA as the reference standard. MethodsFluorescent qPCR was used for initial SMA carrier screening, followed by confirmatory testing with MLPA for all detected carriers. ResultsOf the 10,630 women screened, 219 were identified as carriers (2.06 % detection rate). This included 17 cases of heterozygous deletion of exon 7 (E7), 145 cases with deletions of both E7 and exon 8 (E8), and 57 cases of E8 deletion alone. The carrier rate for E7 heterozygous deletion was established at 1.5 %. Prenatal diagnosis for seven carrier couples revealed five fetuses as carriers and one affected by SMA. The diagnostic concordance between qPCR and MLPA was 100 %. ConclusionThe combined use of qPCR and MLPA is vital in identifying SMA carriers, allowing for early diagnosis and informed reproductive decisions. The high sensitivity and specificity of qPCR, matching MLPA, demonstrate its value in clinical settings for SMA screening and prenatal diagnosis. Our findings emphasize the critical importance of selecting precise diagnostic methods to enhance clinical outcomes in genetic screening programs.

A-272 Evaluation of a Real-Time Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Respiratory Pathogens in Nasopharyngeal Specimens

Abstract Background Respiratory infections present a significant challenge due to the diverse range of pathogens involved and the possibility of concurrent infections, emphasizing the importance of rapid and accurate detection for effective treatment. Advancements in molecular methods have provided cost-effective and reliable solutions for the identification of respiratory pathogens. In this study, we aimed to assess performance characteristics of the Seegene Allplex™ 2019-nCoV Assay and Seegene Novaplex™ Respiratory Panel Assays for simultaneous amplification and detection of 22 respiratory pathogens using real-time multiplex polymerase chain reaction (PCR). Methods Nucleic acid extraction was performed using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, MA) on the CyBio® FeliX liquid handler (Analytik Jena, Germany). PCR amplification and detection were performed using the Seegene Novaplex™ Respiratory Panels 1A, 2, 3, PneumoBacter Assay, and Allplex™ 2019-nCoV Assay (Seegene Inc, South Korea) on the CFX96™ System (Bio-Rad Laboratories, CA). The assays utilize Seegene MuDT™ technology that allows for the detection of multiple targets in a single channel without the need for melt curve analysis. For method validation, precision studies were performed within-run and over 4 days. The limit of detection was verified by using plasmids from Seegene containing the target sequence for each pathogen at known concentrations. Method comparison studies were carried out by extracting and analyzing a range of 41 to 79 split samples for each pathogen against the BioFire® Respiratory 2.1 Panel (bioMérieux, France), cobas® Influenza A/B & RSV Assay on the cobas® liat system (Roche Diagnostics, IN), and Lyra® SARS-CoV-2 Assay (Quidel, CA) on the CFX96™ system. Additional accuracy studies were performed by spiking negative patient samples with standards from ZeptoMetrix® (Antylia Scientific, IL) containing known concentrations of the targeted respiratory pathogens. Results Precision studies demonstrated consistent and reproducible results for all pathogens. The limit of detection was verified at 1000 copies/reaction for human parainfluenza virus 4 and human metapneumovirus, and at 100 copies/reaction for all other pathogens. Results of the spike-and-recovery studies were consistent with expected results for all targets. Patient correlation studies showed 100% agreement with expected pathogen identification for 19 pathogens, and greater than 97.9% agreement for influenza B virus, respiratory syncytial virus, human parainfluenza virus 4, and human coronavirus 229E. Conclusions The Seegene Allplex™ 2019-nCoV Assay and Novaplex™ Respiratory Panel Assays offer a rapid and reliable method for detecting respiratory pathogens in nasopharyngeal specimens. The capability of the assays to detect multiple targets in a single well enhances efficiency, cost-effectiveness, and turnaround time.

B-236 New real-time PCR test for APOE genotyping in patients with Alzheimer’s disease before anti-amyloid therapy

Abstract Background In 2023, the US Food and Drug Administration approved LEQEMBI, a monoclonal antibody therapy targeting beta-amyloid plaques, for patients with early-stage Alzheimer’s disease (AD). However, anti-amyloid drug trials demonstrated an elevated risk for amyloid-related imaging abnormalities with cerebral edema (ARIA-E) in carriers of the AD risk allele apolipoprotein E epsilon 4 (APOE-ɛ4), especially in ɛ4/ɛ4 homozygous individuals [1-3]. Here, we report on the evaluation of a new real-time polymerase chain reaction (PCR) test for APOE genotyping. Methods The EURORealTime APOE (EUROIMMUN), which enables simultaneous testing for all six APOE genotypes (ɛ2/ɛ2, ɛ2/ɛ3, ɛ2/ɛ4, ɛ3/ɛ3, ɛ3/ɛ4, and ɛ4/ɛ4) by real-time PCR, was performed using genomic DNA (gDNA) isolated from EDTA blood samples. The PCR amplification results were evaluated using the EURORealTime Analysis Software (EUROIMMUN). Samples from healthy blood donors (HBD) were used to assess intra-assay, inter-assay and between-lot precision (n=6) as well as analytical sensitivity (n=21), with each APOE genotype represented by at least one sample. Assay accuracy was analyzed by comparing genotype assignments to the results of the CE-IVD-marked EUROArray APOE (EUROIMMUN) and bidirectional Sanger sequencing using 110 samples (100 AD, 10 HBD). APOE genotype frequencies were compared between patients (100 AD) and controls (250 HBD). Results Precision testing of the EURORealTime APOE resulted in 100% correct genotype calls. Assessment of the analytical sensitivity revealed valid and correct genotype assignment in all 21 samples, confirming 1 ng/µl as the recommended minimum gDNA concentration. The analytical accuracy amounted to 100% based on agreement with the two reference methods in 110/110 cases. Comparison of the APOE genotype distribution showed a higher proportion of ɛ4 allele carriers (i.e., ɛ2/ɛ4, ɛ3/ɛ4, and ɛ4/ɛ4) in AD patients than in HBD (50.0% vs. 25.2%). The prevalence of the ɛ4/ɛ4 genotype in these cohorts was 25.0% for AD patients compared with 2.8% for HBD. Conclusions The well-established high prevalence of the APOE-ɛ4/ɛ4 genotype in AD patients, together with the increased risk of ARIA-E in these homozygous carriers, substantiates the relevance of APOE genotyping prior to anti-amyloid treatment. The EURORealTime APOE test provides sensitive and reliable determination of APOE alleles, thus presenting a suitable tool to improve risk stratification for potential LEQEMBI recipients.

Beta-Thalassemia Haplotypes in Southwest of Iran

Background: Thalassemia is a widespread disease affecting people across various ethnicities and regions. In comparison to previous studies conducted in different regions of Iran, such as those in Lorestan and Sistan-Baluchestan, this study highlights unique mutation patterns prevalent in the southwestern population, emphasizing the genetic heterogeneity in this region. The identification of common mutations of beta-thalassemia in various ethnic groups within the nation is regarded as a practical solution for thalassemia prevention and prenatal diagnosis. Materials and Methods: In this retrospective observational study, the medical records of 545 patients with various types of beta-thalassemia (silent, minor, intermediate, and major), referred to the center at Baqaei 2 hospital over a 14-year period (2008–2022), were examined. The age range of patients spanned from a 2-month-old fetus to a 34-year-old individual. Their mutations and thalassemia types were determined and confirmed using molecular methods, including PCR-ARMS (polymerase chain reaction-amplification refractory mutation system) and sequencing. The results were analyzed using SPSS software. Results: The study examined 545 patients and identified 81 types of mutations. The most frequent mutations observed were CD36-37(-T)/N, IVSII-1/N, and IVS1-110(G>A). The study also noted population heterogeneity, reflected in the wide range of mutations found in the region. Among the patients, 6 had the silent form of beta-thalassemia, 488 had the minor form (464 patients and 24 fetuses), 9 had the intermediate form (8 patients and 1 fetus), and 42 had the major form (26 fetuses and 16 adults). Conclusion: The identification of prevalent beta-thalassemia mutations facilitates disease control and prevention programs and is crucial for the identification of various beta-thalassemia gene mutations. This should be re-evaluated periodically. Observing a wide range of beta-thalassemia genotypes in the southwestern region of Iran suggests gene flow; thus, identifying these genotypes is instrumental in preventing and controlling the disease.

Wastewater-based intestinal protozoa monitoring in Shanghai, China.

Intestinal protozoa Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi have been implicated in serious waterborne outbreaks worldwide. Wastewater-based epidemiology (WBE) is a promising approach for evaluating the disease prevalence in a catchment population in that it monitors the contamination level of the intestinal pathogens in wastewater. We collected 48 urban wastewater samples (24 from influents and 24 from effluents) from the Yangpu Wastewater Treatment Plant (YPWTP) in Shanghai, China. We identified Cryptosporidium spp., G. duodenalis, and E. bieneusi by nested polymerase chain reaction (PCR) amplification. Cryptosporidium hominis and subtype IdA14 were identified in two samples by analyzing the sequences of small subunit ribosomal RNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The G. duodenalis sub-assemblage AII (n = 8) and assemblage C (n = 4) in 12 samples were determined by analyzing triosephosphate isomerase (tpi) gene sequences. The E. bieneusi genotype A was identified in one sample by analyzing the sequence of the internal transcribed spacer (ITS) region of the rRNA gene. These findings suggest that improving wastewater treatment and monitoring the virility of pathogens in effluents is critical. We observed similar prevalence and genotypes/subtypes of the three intestinal protozoa in our wastewater samples as those reported in previous studies, providing evidence that WBE can be used as an effective epidemic management tool.IMPORTANCECryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common intestinal protozoa causing diarrhea. The infective oocysts, cysts, and spores released in feces can survive in different environments, including multiple types of water bodies. Humans can acquire these intestinal protozoan infections via the fecal-oral route as in waterborne transmission. Wastewater-based epidemiology can rapidly and reliably detect and monitor the emergence and spread of waterborne diseases. We detected Cryptosporidium spp., G. duodenalis, and E. bieneusi in a wastewater treatment plant in Shanghai, China, reflecting the occurrence and genetic characterizations of the three intestinal pathogens from community members served by the wastewater treatment plant.

The Possible Role of Progesterone Receptors in Prostate Cancer Incidences in the Iraqi Population.

Prostate cancer (PCa) is one of the leading diseases causing mortality. It comes in the third rank of common cancer types. It is considered extremely a complicated cancer type since it occurs in highly steroid-responsive and dependent tissues. Many factors are considered to play an important role in the disease progression of PCa, with some functioning at the molecular level. After applying the exclusion criteria, 200 patients who underwent proctectomy were included in this study. Following receiving patient consent, blood samples were withdrawn from patients, DNA was extracted, and precise polymerase chain reaction (PCR) amplification was conducted using specifically designed primers. The resulting amplicons were sequenced and analyzed. The progesterone receptor B (PGRB) DNA from patients showed four distinctive single-nucleotide polymorphisms (SNPs) at sites 11:101128812, 11:101128924, 11:101128949, and 11:101128986, which altered the amino acid sequences to Y>N, A>D, T>I, and C>R, respectively, compared to control. These SNPs resided in sensitive sites that either affected the control elements or promoted alterations in the protein configuration. This DNA change diminished the PGR gene function and promoted an imbalance in the encoded PGR protein structure and expression. Many factors may play a role in PCa manifestation, with steroids and progesterone initially noted as factors. Many studies have dealt with the hormonal effect on PCa; however, few have ultimately determined the molecular impact on disease progression. The presence of pathogenic SNPs in the enhancing region of the gene may impact the expression level of PGR. High or low expression levels may negatively affect gene function, which can be considered a reliable factor in prostate tumorigenesis.

Occurrence rate and species and subtypes of Cryptosporidium spp. in pet dogs in Yunnan Province, China

BackgroundCryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China.ResultsA total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively.ConclusionsThe present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.

In-vitro activities of essential antimicrobial agents including aztreonam/avibactam, eravacycline, colistin and other comparators against carbapenem-resistant bacteria with different carbapenemase genes: A multi-centre study in China, 2021

Carbapenem-resistant bacteria (CRB), including carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbapenem-resistant Enterobacterales (CRE), pose a considerable threat to public health in China. Eravacycline, aztreonam/avibactam and colistin are important antimicrobial agents for the treatment of serious infections caused by CRB. This study aimed to evaluate the prevalence of CRB strains, and the susceptibility of commonly used clinical antimicrobial agents against strains with different carbapenemase genes. In total, 7194 gram-negative bacteria strains were collected from different regions of China, and 924 carbapenem-resistant strains were identified. All strains were from confirmed infections. Antimicrobial susceptibility testing, covering 21 antimicrobial agents including aztreonam/avibactam, eravacycline, colistin and other comparators, was performed using the broth microdilution method. Carbapenemase genes (blaKPC, blaNDM, blaOXA, blaIMP and blaVIM) were screened using polymerase chain reaction amplification and sequence analysis. All statistical analyses were performed using Statistical Package for the Social Sciences Version 23.0. The isolation rates of CRE, CRAB and CRPA were 6.31% (332/5265), 62.95% (440/699) and 15.20% (152/1000), respectively. The predominant carbapenemase in carbapenem-resistant Escherichia coli (CRECO) was NDM, while in carbapenem-resistant Klebsiella pneumoniae (CRKP), it was KPC. All CRAB produced OXA-23, and 85.52% of CRPA did not produce any of the following carbapenemases: NDM, KPC, VIM, IMP and OXA. Aztreonam/avibactam, colistin and eravacycline exhibited high antimicrobial activity against different species producing various carbapenemases. Compared with ceftazidime/avibactam, aztreonam/avibactam demonstrated superior antimicrobial activity, particularly pronounced in CRECO and strains producing metallo-beta-lactamases. In comparisons between tigecycline and eravacycline, the latter maintained higher antimicrobial activity across different species. Antimicrobial agents exhibited varying levels of activity against strains with different resistance mechanisms. Using aztreonam/avibactam, eravacycline and colistin to treat infections caused by CRB offers significant advantages. These findings will guide clinical practice and optimize antimicrobial administration.

The Factors Associated With Mutant Epidermal Growth Factor Receptor Positivity in Patients With Lung Cancer: A Study From a Tertiary Care Center in Pakistan.

Objective In this study, we sought to elucidate the relationship between demographic and clinical factors and epidermal growth factor receptor tyrosine kinase (EGFR-TK) positivity in patients with advanced-stage lung cancer at a tertiary care center in Pakistan. Methods This analytical cross-sectional study was conducted from February 2020 to July 2023 at Shaikh Zayed Hospital, Lahore, Pakistan, in collaboration with the Centre of Excellence in Molecular Biology (CEMB), University of the Punjab. The study included 70 consecutive patients with advanced-stage lung cancer, and aimed to identify common EGFR mutations (Exon 19 deletion and Exon 21 L858R mutation), determine their frequency, and correlate EGFR-TK mutation positivity with clinical and non-clinical factors. Tissue acquisition and processing involved bronchoscopy, pleuroscopy, or percutaneous ultrasound-guided lung mass biopsy, followed by molecular analysis using polymerase chain reaction (PCR) extraction, amplification, and sequencing. The study employed convenient sampling and included adults aged over 18 years with histologically confirmed lung cancer. Results We enrolled 70 lung cancer patients, with a mean age of 60.87 years, and a male-to-female ratio of 1.4:1. The majority (61.4%) had adenocarcinoma, and 34.3% harbored a mutant EGFR-TK. EGFR mutations were more common in adenocarcinoma (91.7%) and associated with female non-smokers. Binary logistic regression analysis revealed that age <50 years and adenocarcinoma type were significant predictors of EGFR mutations. We found no significant associations with gender, smoking status, or other variables. These findings have implications for personalized treatment approaches in lung cancerpatients. Conclusions Our study reveals a high frequency of occult EGFR mutations (Exon 19 deletion and Exon 21 L858R mutation) in non-small cell lung cancer (NSCLC) patients, particularly among male smokers and female non-smokers. These findings emphasize the importance of routine EGFR mutation testing to identify patients who may benefit from targeted therapies, ultimately improving treatment outcomes.

Olanzapine-induced Metabolic Syndrome and its Association with -759C&gt;T Polymorphism of the HTR2C Gene in Iraqi Schizophrenic Patients

The hazardous metabolic effects of treating schizophrenia patients with olanzapine comprise serotonin 2C receptor (5-HT2C) antagonists. Metabolic side effects of antipsychotic drugs, including lipid abnormalities, disturbed glucose metabolism, and weight gain, can have a major impact on treating psychiatric patients. The intent of this study was to investigate whether there is an associated link between the genetic polymorphism at -759C&gt;T in the promoter region of the 5-hydroxytryptamine 2C receptor (HTR2C) gene and the metabolic syndrome driven by olanzapine in schizophrenia patients. A cross-sectional study that involved fifty hospitalized patients with schizophrenia. The patients were split into two groups (metabolic and non-metabolic) according to the classification criteria of the metabolic syndrome. The HTR2C promoter region polymorphism was identified through sequencing using the Sanger method after polymerase chain reaction amplification of the extracted deoxyribonucleic acid. Even though none of the genotypes of the -759C&gt;T variant are associated with the propensity to develop metabolic syndrome, there is a significant difference in the -759C&gt;T variant's T allele (p-value = 0.001). The presence of the T allele in the -759 C/T variant was significantly associated with developing metabolic syndrome. Keywords: Schizophrenic patients, Olanzapine, Genetic polymorphism, 5-hydroxytryptamine 2C receptor (HTR2C) gene, -759C&gt;T.

A decade-long retrospective study of hepatitis C virus genetic diversity in Cameroon, 2013-2023: presence of a high proportion of unsubtypable and putative recombinant HCV strains.

While treatment options for hepatitis C virus (HCV) infection have expanded considerably over the past decade thanks to the development of pan-genotypic therapies, genotype testing remains a prerequisite for treatment in sub-Saharan African countries, including Cameroon, where multiple HCV genotypes and subtypes exist. The main objective of this study was to describe the trend in the distribution of HCV genotypes and subtypes from 2013 to 2023 in the Cameroonian population. Viral loads were determined using the Abbott real-time assay, and genotyping/subtyping was based on nested and semi-nested reverse transcription polymerase chain reaction (RT-PCR) amplification of the regions encoding the core and non-structural protein 5B (NS5B) regions, respectively, followed by sequencing and phylogenetic analysis. A total of 512 patients with NS5B and core sequencing results were included in our study. Genotyping revealed a predominance of both genotype 4 (38.48%) and genotype 1 (37.11%), followed by genotype 2, detected in 22.46% of patients. Interestingly, 10 samples (1.95%) had discordant genotypes in both regions, suggesting the presence of putative recombinant forms of HCV. Twelve different subtypes were detected during the study period, with a predominance of subtypes 4f (18.95%) and 1e (16.02%). Furthermore, phylogenetic analysis failed to assign a subtype to a relatively high proportion of sequences (38.67%) for the two genomic regions, and their classification was limited to genotype assignment. The frequency distribution of HCV genotypes did not show any statistical difference according to year or sex. These results confirm the genetic diversity of HCV in Cameroon and the potential for the generation of recombinant strains.

Advanced DNA Amplification for Efficient Data Storage.

DNA amplification technologies have significantly advanced biotechnology, particularly in DNA storage. However, adaptation of these technologies to DNA storage poses substantial challenges. Key bottlenecks include achieving high throughput to manage large data sets, ensuring rapid and efficient DNA amplification, and minimizing bias to maintain data fidelity. This perspective begins with an overview of natural and artificial amplification strategies, such as polymerase chain reaction and isothermal amplification, highlighting their respective advantages and limitations. It then explores the prospective applications of these techniques in DNA storage, emphasizing the need to optimize protocols for scalability and robustness in handling diverse digital data. Concurrently, we identify promising avenues, including advancements in enzymatic processes and novel amplification methodologies, poised to mitigate existing constraints and propel the field forward. Ultimately, we provide insights into how to utilize advanced DNA amplification strategies poised to revolutionize the efficiency and feasibility of data storage, ushering in enhanced approaches to data retrieval in the digital age.

Generation and Characterization of a variant of Thermostable Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) Enzyme

Background Reverse Transcriptase (RT) enzymes have been extensively utilized, especially in Polymerase Chain Reaction (PCR). Many viruses that cause infections worldwide contain ribonucleic acid (RNA), and for their molecular detection, it is essential to convert RNA to DNA using RT. This study aimed to create and characterize a thermostable Moloney Murine Reverse Transcriptase (MMLV-RT) enzyme by using consensus sequences from the latest MMLV-RT sequence database. Methodology The six latest sequences encoding MMLV-RT were retrieved from the NCBI website, and the consensus sequence was determined and cloned into the pET28a (+) vector. The vector was overexpressed in an E. coli expression system. The enzyme obtained was purified using Talon accept resin. The purified enzyme was analyzed by SDS-PAGE and Western Blotting. The enzyme performance was evaluated by performing PCR on 89 SARS-CoV 2 positive samples and 40 negative samples both in a concentrated state and at a dilution factor of x10−1. The performance was compared with that of the commercial enzyme, a commercial RT enzyme kit, and Superscript (Invitrogen). Results The enzyme was successfully expressed in E. coli. The concentration of MMLV-RT used was 0.313 mg/mL. The concentrated enzyme detected 98.9% of SARS-CoV-2 RNA, whereas the diluted RT enzyme detected 92.1% of SARS-CoV-2 RNA. In contrast, the diagnostic specificity was 98% for concentrated RT and 95% for diluted RT. This showed that the recombinant in-house MMLV-RT enzyme prototype could be used for the PCR amplification of viral RNA. (Figure 3) Conclusion We successfully produced a recombinant MMLV-RT enzyme whose performance was comparable to that of standard commercial reverse transcriptase (P &lt;0.0001).

PCR-free and minute-scale electrochemical analysis of porcine DNA adulteration via molecularly amplified DNA sandwich assay

The increasing incidence of meat adulteration and mislabeling poses significant challenges in terms of food safety and consumer trust. This study proposes an electrochemical DNA biosensor for detecting porcine mitochondrial DNA in tainted meat products, offering a novel approach to address the above challenges. Unlike conventional nucleic acid amplification tests that rely on polymerase chain reactions (PCRs), the proposed biosensor employs a molecularly amplified DNA strategy with DNA tracers that bind to two regions of the target DNA, creating an elongated hybridization structure with multiple redox-tagging molecules. This design catalyzes detection signals autonomously, eliminating the need for PCR amplification. One-step DNA probe immobilization using poly-adenine (poly-A) oligonucleotides significantly improves hybridization efficiency and reduces the necessity for extensive sample purification, thereby simplifying the detection process. The proposed biosensor exhibits a linear detection range of 101–106 pM and a limit of detection (LOD) of 2.2 pM in controlled settings. Furthermore, the proposed biosensor distinguishes pork from beef in adulterated samples with a LOD of 1 % w/w. With its stability exceeding 9 weeks and a cost of less than 0.5 USD per test, the proposed biosensor offers a highly sensitive, economically viable solution with significant potential for widespread use in the meat industry and by end-users, effectively combating porcine adulteration.