Single Polymerase Research Articles

Redefining copy number variation and single-nucleotide polymorphism counting via novel concepts based on recent PCR enhancements.

Human genes have numerous copy number variations (CNVs) and single-nucleotide polymorphisms (SNPs) that control most of the body’s core functions. On average, 12–16% of human genes have CNVs, and a single gene can have a few hundred to several thousand SNPs. Numerous genome-wide association studies (GWAS) have shown that CNVs and SNPs can coexist in certain genomic regions, amplifying their effects on gene expression and regulation and disease susceptibility. Researchers initially categorized CNVs and SNPs into two types: homozygous and heterozygous. However, copy numbers were soon found to have a much wider range, underscoring their significance in certain diseases and microbial interactions. Because of the significant impact of CNVs and SNPs, research groups worldwide have eagerly sought effective methods for detecting both simultaneously. Despite yielding some minor results, these simultaneous counting methods have failed to meet expectations, leaving researchers to measure CNVs and SNPs separately. To overcome these limitations, we developed a novel approach by combining primers designed using the STexS method with matching probes used in the STexS II method. This method successfully detected both CNVs and SNPs in CYP2A6 and CYP2A7 using a single quantitative polymerase chain reaction. Once properly adjusted based on the three core principles, this new method markedly improved the time, cost-effectiveness, and overall accuracy of determining an individual’s genetic status. Further testing of 100 human genomic DNA samples enabled calculations of the overall frequency of the [T] and [G] alleles of the CYP2A6 -48T>G SNP within an East Asian population yielded results that were highly congruent with those in a National Institutes of Health (NIH) database. This novel method will redefine genetic profiling and provide a means to successfully predict genetic characteristics and enhance personalized medicine by pinpointing appropriate individualized treatments.

Identifying Gaps in the International Consensus Case Definitions for Invasive Aspergillosis: A Review of Clinical Cases Not Meeting These Definitions.

International consensus definitions for invasive aspergillosis (IA) in research are rigorous, yet clinically significant cases are often excluded from clinical studies for not meeting proven/probable IA case definitions. To better understand reasons for the failure to meet criteria for proven/probable infection, we herein review 47 such cases for their clinical and microbiological characteristics and outcomes. Data on 47 cases that did not meet consensus IA definitions but were deemed significant were derived from a retrospective, observational, multicenter survey of 382 presumed IA cases across Australasia, of which findings of 221 proven/probable infections were recently published. The clinical, microbiological, and radiologic characteristics of these cases were analyzed. Mortality outcomes were compared with those of 221 proven/probable cases. Of 47 cases studied, 15 lacked classical host factors; 22 exhibited only a single positive Aspergillus polymerase chain reaction result; 7 lacked typical IA radiologic findings on chest computed tomography; and 3 had borderline galactomannan optical density indices (<1.0 but ≥0.5) in bronchoalveolar lavage fluid. The median age of patients was 61 years (IQR, 52-68); 34 were male (72%). Seven patients (15%) required intensive care admission. All patients had lung as the primary site of infection. Antifungal treatment was initiated in 42 patients (89%). All-cause 90-day mortality was 33%, similar to the 30% mortality in the comparative cohort (n = 221). Our findings highlight the limitations of current consensus definitions for IA. Notably, the mortality of patients not meeting these definitions was similar to that of patients with proven/probable IA. Further studies, especially of patients with a single positive Aspergillus polymerase chain reaction result and those without host factors, are needed to determine if future consensus definitions may benefit from modifications.

DEVELOPMENT OF A RAPID SNP PCR ASSAY TO DIFFERENTIATE BETWEEN EUROPEAN AND NORTH AMERICAN HAPLOTYPES OF ECHINOCOCCUS MULTILOCULARIS.

Herein we describe a single nucleotide polymorphism-specific polymerase chain reaction (PCR) assay to rapidly detect and differentiate variants belonging to the European and North American lineages of Echinococcus multilocularis in clinical samples. This is an extremely relevant and applicable test in North America because the range of E. multilocularis continues to expand across the continent and because of a rise in prevalence in wildlife, domestic animals, and humans. The endemic North American (NA) and introduced European (EU) variants are believed to have different pathogenic potentials, with the EU variants being more infective and pathogenic than the NA variants. The rise of the EU variants of E. multilocularis increases the risk of spillover from wildlife to humans because of its increased potential for infectivity. Current PCR-based diagnostics can detect E. multilocularis deoxyribonucleic acid (DNA), but DNA sequencing is required to identify the specific variant. Our assay provides a straightforward conventional PCR method to differentiate the NA and EU variants, and we suggest this same approach could be used for the diagnosis of other parasites or variants that are genetically very similar. As surveillance continues for E. multilocularis across North America, identifying the different genetic variants from different geographic regions will become essential to understanding the current epidemiological shift that the parasite is experiencing, as well as informing public health decisions in affected areas.

Biochemical characterization of Mycobacterial RNA polymerases.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), perform the essential function of RNA synthesis, we performed a series of in vitro transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.

How does the polymerase of non-segmented negative strand RNA viruses commit to transcription or genome replication?

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.

Charophytic Green Algae Encode Ancestral Polymerase IV/Polymerase V Subunits and a CLSY/DRD1 Homolog.

In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed DNA Methylation requires the activity of the RNA Polymerases IV and V, which produce small RNA precursors and noncoding targets of small RNAs, respectively. These polymerases are distinguished from Polymerase II by multiple plant-specific paralogous subunits. Most RNA-directed DNA Methylation components are present in all land plants, and some have been found in the charophytic green algae, a paraphyletic group that is sister to land plants. However, the evolutionary origin of key RNA-directed DNA Methylation components, including the two largest subunits of Polymerase IV and Polymerase V, remains unclear. Here, we show that multiple lineages of charophytic green algae encode a single-copy precursor of the largest subunits of Polymerase IV and Polymerase V, resolving the two presumed duplications in this gene family. We further demonstrate the presence of a Polymerase V-like C-terminal domain, suggesting that the earliest form of RNA-directed DNA Methylation utilized a single Polymerase V-like polymerase. Finally, we reveal that charophytic green algae encode a single CLSY/DRD1-type chromatin remodeling protein, further supporting the presence of a single specialized polymerase in charophytic green algae.

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction.

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal. Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment. The introduced restriction enzyme cleavage site at the 3' end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3' end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.

Unveilling genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance in Staphylococcus aureus isolated from a Malaysian Teaching Hospital

BackgroundStaphylococcus aureus is a notorious multidrug resistant pathogen prevalent in healthcare facilities worldwide. Unveiling the mechanisms underlying biofilm formation, quorum sensing and antibiotic resistance can help in developing more effective therapy for S. aureus infection. There is a scarcity of literature addressing the genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance among S. aureus isolates from Malaysia.MethodsBiofilm and slime production of 68 methicillin-susceptible S. aureus (MSSA) and 54 methicillin-resistant (MRSA) isolates were determined using a a plate-based crystal violet assay and Congo Red agar method, respectively. The minimum inhibitory concentration values against 14 antibiotics were determined using VITEK® AST-GP67 cards and interpreted according to CLSI-M100 guidelines. Genetic profiling of 11 S. aureus biofilm-associated genes and agr/sar quorum sensing genes was performed using single or multiplex polymerase chain reaction (PCR) assays.ResultsIn this study, 75.9% (n = 41) of MRSA and 83.8% (n = 57) of MSSA isolates showed strong biofilm-forming capabilities. Intermediate slime production was detected in approximately 70% of the isolates. Compared to MSSA, significantly higher resistance of clindamycin, erythromycin, and fluoroquinolones was noted among the MRSA isolates. The presence of intracellular adhesion A (icaA) gene was detected in all S. aureus isolates. All MSSA isolates harbored the laminin-binding protein (eno) gene, while all MRSA isolates harbored intracellular adhesion D (icaD), clumping factors A and B (clfA and clfB) genes. The presence of agrI and elastin-binding protein (ebpS) genes was significantly associated with biofilm production in MSSA and MRSA isolates, respectively. In addition, agrI gene was also significantly correlated with oxacillin, cefoxitin, and fluoroquinolone resistance.ConclusionsThe high prevalence of biofilm and slime production among MSSA and MRSA isolates correlates well with the detection of a high prevalence of biofilm-associated genes and agr quorum sensing system. A significant association of agrI gene was found with cefoxitin, oxacillin, and fluoroquinolone resistance. A more focused approach targeting biofilm-associated and quorum sensing genes is important in developing new surveillance and treatment strategies against S. aureus biofilm infection.

Using a Handful of Transcriptomes to Detect Sex-Linked Markers and Develop Molecular Sexing Assays in a Species with Homomorphic Sex Chromosomes.

To understand the biology of a species, it is often crucial to be able to differentiate males and females. However, many species lack easily identifiable sexually dimorphic traits. In those that possess sex chromosomes, molecular sexing offers a good alternative, and molecular sexing assays can be developed through the comparison of male and female genomic sequences. However, in many nonmodel species, sex chromosomes are poorly differentiated, and identifying sex-linked sequences and developing sexing assays can be challenging. In this study, we highlight a simple transcriptome-based procedure for the detection of sex-linked markers suitable for the development of sexing assays that circumvents limitations of more commonly used approaches. We apply it to the spotted snow skink Carinascincus ocellatus, a viviparous lizard with homomorphic XY chromosomes that has environmentally induced sex reversal. With transcriptomes from three males and three females alone, we identify thousands of putative Y-linked sequences. We confirm linkage through alignment of assembled transcripts to a distantly related lizard genome and readily design multiple single locus polymerase chain reaction primers to sex C. ocellatus and related species. Our approach also facilitates valuable comparisons of sex determining systems on a broad taxonomic scale.

First report on the molecular detection and genetic diversity of Anaplasma marginale in healthy dairy cattle in Khon Kaen province, Thailand

Background and Aim: Bovine anaplasmosis (BA) is one of the most important diseases of ruminants worldwide, causing significant economic losses in the livestock industry due to the high morbidity and mortality in susceptible cattle herds. Anaplasma marginale is the main causative agent of BA occurring worldwide in tropical and subtropical regions. This study aimed to investigate the first molecular detection and genetic diversity of A. marginale in dairy cattle in Khon Kaen Province, Thailand. Materials and Methods: Blood samples were collected from 385 lactating cows from 40 dairy farms in five districts of Khon Kaen, regardless of age and health status. To detect A. marginale, all DNA preparations were used for molecular diagnosis using a single polymerase chain reaction with the msp4 gene target. A phylogenetic tree was constructed from the msp4 gene sequences using molecular genetic characterization. Genetic diversity was calculated as haplotype diversity, haplotype number, number of nucleotide differences, nucleotide diversity, and average number of nucleotide differences. Results: The overall prevalence of A. marginale was 12.72% (49/385). The highest prevalence (17.19%) was found in Ubolratana district, followed by Muang, Kranuan, Khao Suan Kwang, and Nam Phong districts (14.94%, 14.74%, 13.79%, and 3.70%, respectively). Phylogenetic analysis showed that A. marginale was closely related to isolates from Australia (98.96%), China (99.68%), Spain (99.74%), and the USA (99.63%). Conclusion: The molecular prevalence of BA in dairy cattle is the first to be observed in this area, and the genetic variability with separated clusters shown in the msp4 gene of A. marginale revealed species variation in dairy cattle. This significant genetic diversity contributes to the understanding of the diversity of A. marginale and will be important for the control and prevention of A. marginale in dairy cattle. Keywords: Anaplasma marginale, bovine anaplasmosis, dairy cattle, molecular prevalence.

Tetrahedral DNA nanostructures enhance transcription isothermal amplification for multiplex detection of non-coding RNAs

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.

Correlation analysis between polymorphism of leptin and IGFI genes and body measurements in Barki and Farafra sheep

BackgroundGrowth performance, which can be evaluated using various body measurements, is crucial for providing red meat for human consumption. This study aims to improve the growth performance of sheep by identifying the different genotypes of leptin and IGFI genes associated with higher body measurements.MethodsIn this study, we collected blood samples from Barki (n = 30) and Farafra (n = 30) sheep, and measured their body height, width, length, and weight, and tail width and length to assess their growth performance. We used single strand conformation polymorphism polymerase chain reaction (SSCP-PCR) and sequencing analysis to identify single nucleotide polymorphisms (SNPs) and different genotypes of the leptin and insulin-like growth factor receptor (IGFI) genes associated with sheep body measurements.ResultsThe leptin gene (209 bp) and IGFR gene (345 bp) were found to have three (TT, TG, and GG) and (TT, TC, and CC) genotypes, respectively, by SSCP-PCR. Sequence analysis revealed a SNP in the leptin gene (200G > T) with two alleles (G allele: OR058867 and T allele: OR058868). This SNP resulted in the substitution of the amino acid valine with leucine, which increased the width, length, and weight of the body and tail width in the heterozygous form (TG). The IGFI gene had three SNPs: 305 G > T in TT genotype (OR260670), 184C > T, 265T > C, and 305G > T in TC genotype (OR260669), and 265T > C in CC genotype (OR260671). The TC genotype of the IGFR gene was correlated with higher values of body measurements. Barki sheep were found to have the highest values for body weight, width, length, and tail width compared to Farafra sheep.ConclusionWe recommend the use of entire Barki sheep in inbreeding programs to improve growth performance in Egyptian sheep.

Detection of specific RNA targets by multimerization

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to an essential role of RNA in realization of genetic information and diseases development. Here, we report on an approach for detection of RNA targets based on a particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was approved using genetic material of SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate SARS-CoV-2 RNA-positive samples from SARS-CoV-2 negative samples with high reliability. The proposed technique determines detection of RNA even in samples, which undergone multiple freezing.

Detection of Specific RNA Targets by Multimerization.

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to essential role of RNA in transfer of genetic information and development of diseases. Here, we report on an approach for detection of RNA targets based on the particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase, and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was verified by using genetic material of the SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate the SARS-CoV-2 RNA-positive samples from the SARS-CoV-2 negative samples with high reliability. The proposed technique allows detection of RNA even in the samples, which were subjected to multiple freezing-thawing cycles.

Fast genotyping of K-RAS codons 12 and 13 based on streptavidin magnetic microbeads equipped with biotin-coupled single base-pair mismatch PCR (SM-PCR)

In this study, a single base-pair mismatch polymerase chain reaction (SM-PCR) combined with streptavidin magnetic microbeads (MB) for genotyping of codons 12 and 13 of K-RAS gene was established. It was performed by adding three primers including codon 12 forward single-base mismatch primer, codon 13 forward single-base mismatch primer and one universal reverse primer to differentiate single nucleotide polymorphisms. The both forward primers were designed to carry a single-base mismatch nucleotide to either codons 12 and 13. This design was included to increase the primer specificity and to enhance the discrimination power of the point mutation. Two restriction enzymes (StyD4I and HaeIII) were available for double digestion of codons 12 and 13 at the single tube. The wild type of codon 12 and 13 was digested, but the mutant type was not. And then, using the magnetic microbeads for purifying. Finally, wild type of codon 12 and 13 have different emission wavelength, but mutant type did not due to no digestion. This method showed a good consistency between genotyping results and direct sequencing results. The SM-PCR combined with MB method was feasible and efficient for screening of K-RAS codons 12 and 13 in populations.

Ribonucleotide Misincorporation and Reverse Transcriptase Activities of Plasmodium falciparum Apicoplast DNA Polymerase.

Plasmodium falciparumis the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, P. falciparum is characterized by the presence of a unique and essential organelle called the apicoplast. Reminiscent of an algal chloroplast, the apicoplast possesses its own genome, which is maintained by a single apicoplast DNA polymerase (apPol). Ribonucleotides misincorporated into the genome are among the most common lesions encountered by DNA polymerases, and the ability to replicate past these lesions varies widely among characterized enzymes. Here, we have investigated the ribonucleotide (rNTP) misincorporation frequency of apPol and determined its reverse transcriptase (RT) activity across templating ribonucleotides. Pre-steady-state kinetic experiments indicate that apPol does not have an unusually high discrimination between deoxy and ribonucleotides, with frequencies ranging between 104 and 106 depending on the identity of the ribonucleotide. Once incorporated into its template, apPol can replicate across ribonucleotides using its RT activity, but extension of a deoxynucleotide basepaired with the ribonucleotide is slow relative to a canonical basepair. Exonuclease assays indicate that apPol proofreads ribonucleotides an order of magnitude faster than extension, suggesting that most, but not all, misincorporated ribonucleotides will be excised. Although the components have not been identified, ribonucleotide excision repair or other tolerance mechanisms may exist in the P. falciparum apicoplast, and more targeted proteomic efforts will be needed to elucidate them.

Transient State Kinetics of Plasmodium falciparum Apicoplast DNA Polymerase Suggests the Involvement of Accessory Factors for Efficient and Accurate DNA Synthesis.

Plasmodium, the causative agent of malaria, belongs to the phylum Apicomplexa. Most apicomplexans, including Plasmodium, contain an essential nonphotosynthetic plastid called the apicoplast that harbors its own genome that is replicated by a dedicated organellar replisome. This replisome employs a single DNA polymerase (apPol), which is expected to perform both replicative and translesion synthesis. Unlike other replicative polymerases, no processivity factor for apPol has been identified. While preliminary structural and biochemical studies have provided an overall characterization of apPol, the kinetic mechanism of apPol's activity remains unknown. We have used transient state methods to determine the kinetics of replicative and translesion synthesis by apPol and show that apPol has low processivity and efficiency while copying undamaged DNA. Moreover, while apPol can bypass oxidatively damaged lesions, the bypass is error-prone. Taken together, our results raise the following question─how does a polymerase with low processivity, efficiency, and fidelity (for translesion synthesis) faithfully replicate the apicoplast organellar DNA within the hostile environment of the human host? We hypothesize that interactions with putative components of the apicoplast replisome and/or an as-yet-undiscovered processivity factor transform apPol into an efficient and accurate enzyme.

Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination.

HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction(PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

The molecular analysis of antibiotic resistance and identification of the aerobic bacteria isolated from pleural fluids obtained from patients.

Pleural effusion is a common clinical condition due to various etiological causes. Infectious pleural effusion can be seen in 20-40% of patients. In this study, follow-up of patients admitted to our hospital and diagnosed with pleural effusion are reported. It was aimed to investigate the prevalence of bacteria isolated from patients with pleural effusion and their antibiotic resistance profiles. The pleural fluids obtained from the patients during surgical operations were analyzed microbiologically. Conventional culture, Vitek 2, 16S rRNA, and single Polymerase Chain Reaction (sPCR) were used for microbiological analysis. Twenty-two (12.2%) bacteria were isolated from 180 patients. The most prominent of them were 16 (8.8%) Klebsiella pneumoniae strains. As for the antibiotic sensitivity, gram-negative bacteria showed the highest sensitivity to colistin, while Gram-positive bacteria showed sensitivity to different antibiotics. In 16S rRNA PCR, 22 samples were found to be positive. In the analysis of antibiotic resistance genes, the OXA-48 gene was determined as the highest. In our region, it is essential to perform a microbiological analysis of the sample in patients with pleural effusion. It was thought that revealing both the phenotype and genotype of the antibiotic resistance of the patients was important in terms of treatment. In hospital surveillance, it was considered important to reveal and record the resistance gene profiles of the patients.

Chimeric DNA byproducts in strand displacement amplification using the T7 replisome.

Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation.