Primer Sequences Research Articles

Evaluating the efficiency of 16S-ITS-23S operon sequencing for species level resolution in microbial communities

Rapid advancements in long-read sequencing have facilitated species-level microbial profiling through full-length 16S rRNA sequencing (~ 1500 bp), and more notably, by the newer 16S-ITS-23S ribosomal RNA operon (RRN) sequencing (~ 4500 bp). RRN sequencing is emerging as a superior method for species resolution, exceeding the capabilities of short-read and full-length 16S rRNA sequencing. However, being in its early stages of development, RRN sequencing has several underexplored or understudied elements, highlighting the need for a critical and thorough examination of its methodologies. Key areas that require detailed analysis include understanding how primer pairs, sequencing platforms, and classifiers and databases affect the accuracy of species resolution achieved through RRN sequencing. Our study addresses these gaps by evaluating the effect of primer pairs using four RRN primer combinations, and that of sequencing platforms by employing PacBio and Oxford Nanopore Technologies (ONT) systems. Furthermore, two classification methods (Minimap2 and OTU clustering), in combination with four RRN reference databases (MIrROR, rrnDB, and two versions of GROND) were compared to identify consistent and accurate classification methods with RRN sequencing. Here we demonstrate that RRN primer pair choice and sequencing platform do not substantially bias taxonomic profiles for most of the tested mock communities, while classification methods significantly impact the accuracy of species-level assignments. Of the classification methods tested, Minimap2 classifier in combination with the GROND database most consistently provided accurate species-level classification across the communities tested, irrespective of sequencing platform.

Single-cell Rapid Capture Hybridization sequencing to reliably detect isoform usage and coding mutations in targeted genes.

Single-cell long-read sequencing has transformed our understanding of isoform usage and the mutation heterogeneity between cells. Despite unbiased in-depth analysis, the low sequencing throughput often results in insufficient read coverage thereby limiting our ability to perform mutation calling for specific genes. Here, we developed a single-cell Rapid Capture Hybridization sequencing (scRaCH-seq) method that demonstrated high specificity and efficiency in capturing targeted transcripts using long-read sequencing, allowing an in-depth analysis of mutation status and transcript usage for genes of interest. The method includes creating a probe panel for transcript capture, using barcoded primers for pooling and efficient sequencing via Oxford Nanopore Technologies platforms. scRaCH-seq is applicable to stored and indexed single-cell cDNA which allows analysis to be combined with existing short-read RNA-seq datasets. In our investigation of BTK and SF3B1 genes in samples from patients with chronic lymphocytic leukaemia (CLL), we detected SF3B1 isoforms and mutations with high sensitivity. Integration with short-read scRNA-seq data revealed significant gene expression differences in SF3B1-mutated CLL cells, though it did not impact the sensitivity of the anti-cancer drug venetoclax. scRaCH-seq's capability to study long-read transcripts of multiple genes makes it a powerful tool for single-cell genomics.

DNA Methylation Analysis by Bisulfite Pyrosequencing of Mouse Embryonic Fibroblasts with Reprogramming Enhanced by Thyroid Hormones.

DNA methylation is a widely studied epigenetic mark which in mammals involves the incorporation of a methyl group to the fifth carbon of cytosines, mainly those belonging to CpG dinucleotides. It has been linked to context-dependent regulatory functions ranging from gene and repetitive DNA silencing to gene body transcriptional activity. Because of its important roles during embryonic development and cell differentiation, DNA methylation can be used to track cell reprogramming by measuring the methylation levels of pluripotency-associated factors. In this scenario, bisulfite pyrosequencing is a simple, robust, and widely used technique which allows for the quantification of DNA methylation levels at small, specific regions of the genome. It involves the amplification and biotin tagging of bisulfite-converted DNA. Single amplified strands are then purified using streptavidin and finally pyrosequenced using a sequencing primer. Thus, it is an ideal method for the quantitative profiling of specific genomic regions, with applications ranging from biomarker discovery and epigenetic clock tracking to omic validation studies.

DNA lesion-gated dumbbell nanodevices enable on-demand activation of the cGAS-STING pathway for enhancing cancer immunotherapy.

Utilizing the cGAS-STING pathway to combat immune evasion is one of the most promising strategies for enhancing cancer immunotherapy. However, current techniques for activating the cGAS-STING pathway often face a dilemma, mainly due to the balance between efficacy and safety. Here, we develop a uracil base lesion-gated dumbbell DNA nanodevice (UBLE) that allows on-demand activation and termination of the cGAS-STING pathway in tumor cells, thereby enhancing cancer immunotherapy. The UBLE integrates two deoxyuridines (dU) in the stem for DNA lesion recognition, two locked complementary primer sequences (primers A and B) for DNA self-assembly, and a Förster resonance energy transfer pair (Cy3 and Cy5) attached to the loop for activation assessment. Upon the orthogonal recognition of tumor-specific repair indicators (UDG and APE1), the UBLE undergoes a conformational change to create massive nicked double-stranded DNA (dsDNA) units. These units self-assemble to generate long fluorescent dsDNA structures, permitting selective evaluation and on-demand activation of the cGAS-STING pathway. Furthermore, we demonstrate that the UBLE can effectively activate the cGAS-STING pathway in tumor cells, enhancing NK cell-targeted cancer immunotherapy. This work develops a DNA lesion-gated strategy for on-demand activation and termination of the cGAS-STING pathway, affording an innovative avenue for enhancing cancer immunotherapy.

Machine learning-optimized targeted detection of alternative splicing.

RNA sequencing (RNA-seq) is widely adopted for transcriptome analysis but has inherent biases that hinder the comprehensive detection and quantification of alternative splicing. To address this, we present an efficient targeted RNA-seq method that greatly enriches for splicing-informative junction-spanning reads. Local splicing variation sequencing (LSV-seq) utilizes multiplexed reverse transcription from highly scalable pools of primers anchored near splicing events of interest. Primers are designed using Optimal Prime, a novel machine learning algorithm trained on the performance of thousands of primer sequences. In experimental benchmarks, LSV-seq achieves high on-target capture rates and concordance with RNA-seq, while requiring significantly lower sequencing depth. Leveraging deep learning splicing code predictions, we used LSV-seq to target events with low coverage in GTExRNA-seq data and newly discover hundreds of tissue-specific splicing events. Our results demonstrate the ability of LSV-seq to quantify splicing of events of interest at high-throughput and with exceptional sensitivity.

Loop-mediated isothermal amplification assays for the detection of antimicrobial resistance elements in Vibrio cholera

BackgroundThe bacterium Vibrio cholerae causes diarrheal illness and can acquire genetic material leading to multiple drug resistance (MDR). Rapid detection of resistance-conferring mobile genetic elements helps avoid the prescription of ineffective antibiotics for specific strains. Colorimetric loop-mediated isothermal amplification (LAMP) assays provide a rapid and cost-effective means for detection at point-of-care since they do not require specialized equipment, require limited expertise to perform, and can take less than 30 min to perform in resource limited regions. LAMP output is a color change that can be viewed by eye, but it can be difficult to design primer sets, determine target specificity, and interpret subjective color changes.MethodsWe developed an algorithm for the in silico design and evaluation of LAMP assays within the open-source PCR Signature Erosion Tool (PSET) and a computer vision application for the quantitative analysis of colorimetric outputs. First, Primer3 calculates LAMP primer sequence candidates with settings based on GC-content optimization. Next, PSET aligns the primer sequences of each assay against large sequence databases to calculate sufficient sequence similarity, coverage, and primer arrangement to the intended taxa, ultimately generating a confusion matrix. Finally, we tested assay candidates in the laboratory against synthetic constructs.ResultsAs an example, we generated new LAMP assays targeting drug resistance in V. cholerae and evaluated existing ones from the literature based on in silico target specificity and in vitro testing. Improvements in the design and testing of LAMP assays, with heightened target specificity and a simple analysis platform, increase utility for in-field applications. Overall, 9 of the 16 tested LAMP assays had positive signal through visual and computer vision-based detection methods developed here. Here we show LAMP assays tested on synthetic AMR gene targets for aph(6), varG, floR, qnrVC5, and almG, which allow for resistance to aminoglycosides, penicillins, carbapenems, phenicols, fluoroquinolones, and polymyxins respectively.

Bioinformatic Comparisons of Some Web-based PCR Primer Design Programs

Bioinformatics has become an indispensable tool for both basic and applied research in biotechnology in the life sciences. The polymerase chain reaction (PCR) is a laboratory method that can be used to quickly amplify a large number of identical copies of a specific DNA segment. In PCR, short synthetic DNA fragments known as primers are used to selectively amplify a specific section of the genome. For PCR to be as efficient and specific as possible, it is important to choose an effective primer sequence and use the correct concentration of primers. If the primer is not designed carefully, non-specific amplification and/or primer dimer formation may occur, which may prevent product formation. Currently, a number of different design tools are available on the internet to assist molecular geneticists in designing PCR primers under optimal conditions. In this study, out of 39 web-based PCR primer design programs, 7 accessible, freely available and widely used web-based PCR primer design programs (NCBI, Primer3, Biserach, Genscript and Primer3plus; Stitcher 2.0; and PrimerQest Tool) were compared using bioinformatics applications for genomic sequences. The advantages and disadvantages of the web-based PCR programs are discussed on the basis of the comparison results.

Clade-specific long-read sequencing increases the accuracy and specificity of the gyrB phylogenetic marker gene.

Previous studies have shown that the marker gene gyrase subunit B (gyrB) can be used to study codiversification between the gut microbiome and hominids. However, only primers for short-read sequencing have been developed which have limited resolution for subspecies assignment. In the present study, we create new gyrB primer sets for long-read sequencing approaches and compare them to the existing short-read gyrB primers. We show that using longer reads leads to better taxonomic resolution, lower off-target amplification, and lower error rates, which are vital for accurate estimates of codiversification.

Customised 96-ocular TaqMan (iCAM) microarray PCR card for rapid diagnosis of microbial keratitis

AimTo validate the diagnostic performance of a custom 96-micro-organism TaqMan PCR card (iCAM) for microbial keratitis (MK) from a single corneal epithelial sample.MethodsPatients over the age of 18 referred to...

Nuclear eDNA metabarcoding primers for anthozoan coral biodiversity assessment.

The distributions of anthozoan corals are undercharacterized due to their wide bathymetric ranges, occurrences in remote locales, and difficulties of identification from morphology alone. Environmental DNA (eDNA) sequencing promises to be a noninvasive strategy to complement conventional approaches for mapping and monitoring the distribution and biodiversity of coral communities. Primers for eDNA metabarcoding have been designed to amplify nuclear and mitochondrial DNA barcodes in shallow scleractinians and mitochondrial MutS in deep-sea octocorals. However, a comprehensive method for eDNA metabarcoding of all anthozoan corals, including black corals, has not been developed. We leveraged a sequence database of global coral collections, from shallow water to the deep sea, to design new PCR primers for coral eDNA sequencing that target the 28S rRNA gene (28S rDNA). We tested the performance of these primers by amplifying and sequencing eDNA from water samples collected in the Gulf of Mexico near mesophotic and deep-sea corals that were also imaged, sampled, and sequenced. Sequencing libraries produced using the primers were highly enriched in eDNA from octocorals, black corals and scleractinians, with up to 99.9% of the reads originating from these corals. Further, the 28S barcode amplified using the primers distinguished coral genera and species in many cases, like previously developed methods that target eDNA in only octocorals or scleractinians. We recovered amplicon sequencing variants (ASVs) identical to DNA barcodes derived from Sanger sequencing and genome skimming of corals sampled at the same field sites. This new eDNA metabarcoding strategy permits targeted eDNA sequencing of black corals, octocorals, and scleractinians at sites where they co-occur and expands our current toolkit for mapping and monitoring coral communities in shallow coral reefs and the deep sea.

Analysis of BRCA1 Exon 2 Mutations in FFPE Tissues of Bangladeshi Bangali Females with Breast Cancer

Background: Among all breast cancers, 5 to 10% are hereditary and related to mutation of BRCA1 and BRCA2 gene. To date, extensive researches have been carried out for detection of mutations in the exon2 of BRCA1 gene. Genetic study on these two genes from formalin-fixed paraffin-embedded tissue in Bangladeshi population has not been reported so far. Objective: To identify mutation in exon2 of BRCA1 gene from formalin-fixed paraffin-embedded tissue of adult Bangladeshi Bangali female patients with breast cancer. Methods: The research was cross-sectional descriptive type of study. The study was conducted on Adult Bangladeshi Bangali female patients with breast cancer aged more than 18 years. A questionnaire was developed to record information on socio-demographic and reproductive characteristics. The data was analyzed by SPSS (Statistical package for the social sciences) version 23.0. The whole sequence of exon2 of chromosome 17 was amplified by short range PCR using the Gotaq master mix and primer sequence. The amplicons were confirmed by 2% agarose gel electrophoresis and sequencing was done by Sanger sequencer. Data analysis was performed using Chromas® software version 2.33 and Mega 7 software. The quary sequences were compared with the NCBI database. Results: Average age of the breast cancer patients was 45.46 (± 11.52) and the mean age at menarche was 12.46 (± 0.646). In this study, new mutations along with known mutations were found in exon2 of BRCA1 gene of eight adult female breast cancer patients. The mutations are insertions (C>A, C>T, T>A, G>T, G>C, T>C, A>T, C>G), deletion (C_) and single nucleotide substitution (T-C, G-A, T-G, C-G, T-A, G-C, C-A) in types. Conclusions: The aim of the research was to identify mutation in exon2 of BRCA1 gene. Eight patients have mutation in their DNA sequence revealed from formalin-fixed paraffin-embedded tissue. Further studies are required to evaluate whether these mutation contribute to breast cancer or not. Identification of known mutation along with new mutation with this small sample size emphasizes the importance of exploration the genetic makeup of Bangladeshi population to develop a database for proper screening and genetic counseling of the disease.

Retracted] lncRNA DQ786243 promotes hepatocellular carcinoma cell invasion and proliferation by regulating the miR‑15b‑5p/Wnt3A axis.

Following the publication of this paper, and subsequently to the publication of a corrigendum (DOI: 10.3892/mmr.2022.12912) that was intended to address the issue of a pair of incorrectly written primer sequences, it was drawn to the Editors' attention by a concerned reader that certain of the cell invasion assay data shown in Fig. 4A on p. 7 were strikingly similar to data appearing in different form in an article written by different authors at different research institutes that had already been published in the journal Journal of Cancer. In view of the fact that the abovementioned data had already apparently been published prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 23: 318, 2021; DOI: 10.3892/mmr.2021.11957].

Comparative proteomic and genomic analysis of Lactococcus garvieae and Lactococcus petauri isolates from Turkey

Piscine lactococcosis, a bacterial disease caused by Lactococcus garvieae, L. petauri and L. formosensis, induces mass mortalities in multiple farmed fish species globally. Following the identification of L. petauri as one of the emerging etiological agents of piscine lactococcosis, it was revealed that the majority of isolates previously recognized as L. garvieae actually belong to L. petauri. In this study, a total of 134 Lactococcus sp. isolates, including L. garvieae (n = 40) and L. petauri (n = 94) were analyzed. Differential identification of L. garvieae and L. petauri isolates was performed through basic sucrose fermentation and end-point PCR analysis using species-specific primer sequences. Comparative analysis of capsule formation in both species was conducted for the first time using transmission electron microscopy, whole-cell protein profile via 1D SDS-PAGE, antigenic protein characterization using Western blot analysis, and serological profile description by slide and microagglutination assays. Moreover, comprehensive genomic comparisons were made, deciphering overall genomic relatedness indices, virulence genes, and antimicrobial resistance patterns among these closely related species.Analysis showed microcapsule formation in some isolates of L. garvieae and L. petauri for the first time. While a high similarity rate was observed in the whole-cell protein profiles of these species, significant differences were identified among antigenic proteins. Variations between species were observed in slide agglutination and quantitative agglutination tests of L. garvieae and L. petauri field isolates. Capsule-deficient L. garvieae reference and field strain antisera did not agglutinate with each other; however, L. garvieae field strain antisera agglutinated with all L. garvieae field strains and L. petauri strains. These results indicated distinct serological characteristics of capsule-deficient L. garvieae isolates from Turkey compared to previously reported serological classifications.Genomic analyses revealed that none of the investigated L. garvieae and L. petauri isolates harbored the capsule gene cluster. In addition, Turkish isolates of L. garvieae and L. petauri clustered separately from isolates of other geographical origins but together with each other. This study aims to address the problem of lactococcosis, commonly referred to as “Summer Mortality Outbreak and Vaccine-Preventable Diseases,” in a wide variety of cultured fish species through the selected L. garvieae and L. petauri isolates as potential vaccine strains.

Genetic screening of α-thalassemia fusion gene using routine flow-through hybridization.

The fusion gene is a rare form of α-thalassemia. Patients carrying the fusion gene could be misdiagnosed as normal or -α4.2deletion by the conventional thalassemia detection methods. The aim of this study was to present the detection of fusion genes using routine flow-through hybridization, as well as to analyze hematological and molecular characteristics. Samples were collected at our hospital from January 2019 to January 2024. Common thalassemia mutations in the Chinese population were conducted by flow-through hybridization. Samples showing faint coloration at the -α4.2 mutation site on hybridization membrane were considered suspicious. Samples detected as suspicious for -α4.2deletion were rechecked by conventional Gap-PCR. Those samples suspected of having -α4.2deletions were finally confirmed with specific primers for Gap-PCR and Sanger sequencing. Of the 32,083 samples, 25 samples (0.08%) were detected as suspected of having -α4.2 deletion by flow-through hybridization. However, upon reevaluation wtih conventional Gap-PCR reagents capable of detecting -α4.2 deletion, all were found to be negative for the deletion. Specific primers for Gap-PCR were designed, and fusion gene fragments were amplified. DNA sequencing of the HBA gene showed a 7-base mutation corresponding to the α-thalassemia fusion gene. Among the 25 samples, 22 were heterozygous carriers. Three samples were combined: one with Hb QS, one with β-thalassemia, and one with Hb G-Honolulu.Most hematological indices and capillary electrophoresis results were in the normal reference range. The fusion gene was present in 0.08% of the population in the Guangzhou region of Guangdong province, southern China. Conventional genetic methods tend to misdiagnose the fusion gene but can be effectively screened with flow-through hybridization.

A novel, rapid, ultrasensitive diagnosis platform for detecting Candida albicans using restriction endonuclease-mediated real-time loop-mediated isothermal amplification.

Candida albicans (C. albicans, CA) is an essential invasive fungus in clinical diagnosis. Although several detection methods exist, none meet the need for early diagnosis. A rapid, sensitive, and specific diagnostic tool is crucial for effective prevention and control of C. albicans infections. This study aimed to develop a new, rapid, and ultrasensitive diagnostic tool for C. albicans detection based on restriction endonuclease-mediated real-time loop-mediated isothermal amplification (ERT-LAMP-CA). The ERT-LAMP-CA technology combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence detection in a single reaction tube, which can complete a diagnosis of C. albicans in a short time (approximately 1 h). Herein, we developed the primer sequences required for ERT-LAMP-CA based on the ITS2 gene of C. albicans and found that ERT-LAMP-CA limit of detection was approximately 500 ag/μL genomic DNA and can present negative results for non-C. albicans templates. We tested sputum samples from 64 patients with suspected C. albicans infections to validate ERT-LAMP-CA applicability in clinical sample testing and found that ERT-LAMP-CA was consistent with multiplex PCR-capillary electrophoresis. In conclusion, ERT-LAMP-CA is a rapid, accurate, and sensitive assay with excellent potential for clinical and basic laboratory diagnosis and an efficient screening strategy.

ViralPrimer: a web server to monitor viral nucleic acid amplification tests' primer efficiency during pandemics, with emphasis on SARS-CoV-2 and Mpox.

Accurate pathogen identification is crucial during outbreaks, especially with the emergence of new variants requiring frequent primer updates. However, resources for maintaining up-to-date verification of primer sequences are often limited, which poses challenges for reliable diagnosis and hinders potential monitoring efforts based on genome sequencing. To address this, we introduce ViralPrimer, a web server facilitating primer design, SARS-CoV-2 and Mpox variant monitoring, and adaptation to future threats. ViralPrimer aims to enhance diagnostic accuracy with its comprehensive primer database, mutation analysis assistance, and user primer upload feature. Its adaptable design allows monitoring of other rapidly mutating pathogens, contributing to broader public health protection efforts. ViralPrimer is freely accessible and open to all users with no login requirement at https://viralprimer.elte.hu/. The application is hosted on a DJANGO v3.2.13 web server, with a PostgreSQL database, and the frontend was implemented using jQuery v3.6.0, vanilla JavaScript vES6, and Bootstrap v5.1.

Improved costs and turnaround time for Treponema pallidum detection utilising a real-time PCR assay developed for the Hologic Panther Fusion system

The aims of this study were to evaluate the performance and workflow characteristics of a laboratory-developed test to detect Treponema pallidum on the Hologic Panther Fusion system compared with an existing commercial assay. A Hologic Panther Fusion–based real-time polymerase chain reaction assay was optimised for T. pallidum (TP RT-PCR) using previously published primer and probe sequences and validated with a simplified preprocessing protocol. A total of 124 clinical and external quality assurance samples were tested in parallel by the new method and the Viasure T. pallidum RT-PCR (Certest Biotech). Cross reactivity, limit of detection, PCR efficiency and coefficient of variation (CV) were measured. Simplified preprocessing methods involving either concentration or vortexing were compared with the existing overnight chemical/enzymatic preprocessing method for a subset of positive samples. Workflow impacts were assessed before and after implementation. The Panther Fusion T. pallidum assay correctly detected 65 of 65 (100%) positive clinical and EQA samples and 33 of 33 (100%) negative samples. No cross reactivity was observed for 45 of 45 (100%) samples. The limit of detection was 15 copies/reaction, and intrarun CV was <1.66%. The simplest preprocessing protocol detected 34 of 34 (100%) positive samples with an average ΔCt <0.82 compared with the Viasure workflow. The new workflow reduced median turnaround time from 3.83 to 1.73 days (p < 0.001) and estimated costs from US$35,004.96 to US$19,390.15 in the 3 months postimplementation. The laboratory-developed Panther Fusion T. pallidum assay is a sensitive and specific method for detection of syphilis and a cost-effective option to help improve workflow and turnaround time in a diagnostic laboratory.

An Economic and Reliable PCR Approach to Distinguish the Primary Pathogens Armillaria mellea and A. ostoyae From Other European Armillaria Species

ABSTRACTArmillaria is a soil‐borne genus of basidiomycetes whose species can cause stem and root rot in woody plants. The effects of plant‐pathogenic Armillaria species are well known in forests, but are underestimated in urban areas, where cases causing damage to trees and shrubs in green spaces have been steadily increasing in Switzerland since the 1980s. In this study, we present a simple, rapid, and cost‐effective protocol for high‐throughput diagnostics of the two primary pathogens A. mellea and A. ostoyae based on partial PCR amplification of the RPB2 gene. The specificity and sensitivity of the presented duplex PCR‐I and single‐plex PCR‐II were evaluated using different methods: (i) testing both PCRs on tree pathogenic or soil‐borne fungi of genera other than Armillaria, (ii) using dilution series of Armillaria‐DNA to determine a minimum detection limit, and (iii) sequencing the selected RPB2 region to verify the primer sequences and positions. The utility of PCR‐I and PCR‐II as a high‐throughput method was successfully tested on 65 DNA samples of Armillaria from Switzerland. Finally, an uninvolved person compared both classical methods, pairing test and sequencing, with PCR‐I and PCR‐II in a blind test. This study provides a reliable and alternative protocol for the rapid diagnosis of A. mellea and A. ostoyae causing root rot of woody plants.

Development of novel digital PCR assays for the rapid quantification of Gram-negative bacteria biomarkers using RUCS algorithm

Rapid and accurate identification of bacterial pathogens is crucial for effective treatment and infection control, particularly in hospital settings. Conventional methods like culture techniques and MALDI-TOF mass spectrometry are often time-consuming and less sensitive. This study addresses the need for faster and more precise diagnostic methods by developing novel digital PCR (dPCR) assays for the rapid quantification of biomarkers from three Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.Utilizing publicly available genomes and the rapid identification of PCR primers for unique core sequences or RUCS algorithm, we designed highly specific dPCR assays. These assays were validated using synthetic DNA, bacterial genomic DNA, and DNA extracted from clinical samples. The developed dPCR methods demonstrated wide linearity, a low limit of detection (∼30 copies per reaction), and robust analytical performance with measurement uncertainty below 25 %. The assays showed high repeatability and intermediate precision, with no cross-reactivity observed. Comparison with MALDI-TOF mass spectrometry revealed substantial concordance, highlighting the methods’ suitability for clinical diagnostics.This study underscores the potential of dPCR for rapid and precise quantification of Gram-negative bacterial biomarkers. The developed methods offer significant improvements over existing techniques, providing faster, more accurate, and SI-traceable measurements. These advancements could enhance clinical diagnostics and infection control practices.

Splicing by Overlap Extension PCR for the Production of Fusion Proteins.

Fusion proteins are valuable molecules to meet different demands related to the development of biopharmaceuticals and bioprocesses. In human therapy, they are used to improve the half-life of biologics by modifying the biophysical properties of the proteins. In biotechnology, the design of fusion proteins can standardize the establishment of production clones and the purification process. Analytical detection capabilities of the fusion partner and binding to affinity ligands play a crucial role. For the generation of the recombinant cell line, the maturation of the protein and the secretion are also crucial factors, which can be significantly influenced by the fusion partner and candetermine the final yield of the bioprocess. Here we present a protocol to recombine the human extracellular domain of CD19 with the human serum albumin domain 2 resulting in a fusion protein CD19-AD2 including a flexible linker sequence in the interface between the C-terminus of CD19 and the N-terminus of HSA-D2 and a terminal His12-tag. The two fragments are independently amplified with primers allowing to genetically connect the two fragments in the next step by overlap extension PCR. By this strategy, the linker sequence as well as the albumin fragment can be chosen individually to be flexible in the fine-tuning of the final protein. The amplified product is then cloned into a mammalian expression vector suitable to generate a recombinant transient or stable cell culture. This workflow can be applied to any protein sequence by adapting the specific primer sequences.