Nucleic Acid Analysis Research Articles

Development of metrological support for nucleic acid measurements

Measurements of nucleic acid have become an essential part of analytical applications in many fields including life science, medicine, veterinary medicine and biotechnology. Peculiarities of these measurements are largely determined by the uniqueness of quantity and analyte definitions. Quantitative PCR, applied for routine nucleic acids measurements, is a relative method demanding external calibrators with quantity values traceable to corresponding reference materials. Direct nucleic acid measurements became possible after introduction of the digital PCR (dPCR) method. Ensuring accurate, comparable and traceable to SI nucleic acids measurements is the focus of Nucleic Acid Analysis Working Group of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology. A number of pilot and key comparisons carried out by Working Group have shown that application of dPCR allows participating National Metrological Institutes to obtain comparable results of measurements of the sequence copy number concentrations and copy number ratios.

Evaluation of the BioFire FilmArray Pneumonia Panel Plus to the Conventional Diagnostic Methods in Determining the Microbiological Etiology of Hospital-Acquired Pneumonia.

Simple SummaryHospital-acquired pneumonia (HAP) imposes public health threats because of its high morbidity and mortality rate. Accordingly, this study aimed to evaluate the diagnostic performance of the multiplexed BioFire FilmArray Pneumonia Panel plus (BFPP) for the rapid detection of various clinically relevant respiratory pathogens and genetic markers among 50 patients admitted with HAP to an intensive care unit (ICU) in a tertiary care hospital in Egypt. In comparison to standard culture methods, BFPP showed an overall sensitivity and specificity of 100% and 90%, respectively, with the identification of 11 viral targets (22%) among the tested specimens. The BFPP semi-quantitative analysis showed a concordance rate of 47.4% among positive culture specimens. For the examination of the antibiotic resistance genes, BFPP showed a positive percent agreement (PPA), a negative percent agreement (NPA) and an overall percent agreement (OPA), reaching 97%, 95%, and 95%, respectively, with standard antibiotic sensitivity testing. According to the obtained results, BFPP has the potential to enhance the rapid microbiological diagnosis of HAP cases, tailor appropriate antibiotic therapy, apply antimicrobial stewardship programs, and implement effective infection control measures.Hospital-acquired pneumonia (HAP) is a substantial public health issue that is associated with high mortality rates and is complicated by an arsenal of microbial etiologies, expressing multidrug-resistant phenotypes, rendering relatively limited therapeutic options. BioFire FilmArray Pneumonia Panel plus (BFPP) is a simple multiplexed PCR system that integrates sample preparation, nucleic acid extraction, amplification, and analysis of microbial etiology, with a turnaround time of about one hour. In comparison to standard culture methods, BFPP is simpler, easier to perform, and can simultaneously detect the most common pathogens involved in lower respiratory tract infections (34 targets). Accordingly, we evaluated the diagnostic performance of the multiplexed BFPP for the rapid detection of 27 clinically relevant respiratory pathogens and 7 genetic markers among 50 HAP cases admitted to the intensive care unit (ICU), who submitted mini-bronchoalveolar (mBAL) specimens. In comparison to standard culture methods, BFPP showed an overall sensitivity of 100% [95% CI; 90–100] and overall specificity of 90% [95% CI; 87.4–92.5] among all the tested bacterial targets. BFPP identified 11 viral targets (22%) among the tested specimens. The BFPP semi-quantitative analysis showed a concordance rate of 47.4% among positive culture specimens. For the investigation of the antibiotic resistance genes, BFPP showed a positive percent agreement (PPA), a negative percent agreement (NPA), and an overall percent agreement (OPA), reaching 97% [95% CI; 90–100], 95% [95% CI; 91.5–97], and 95% [95% CI; 93–97], respectively, with standard antibiotic sensitivity testing. In conclusion, BFPP has the potential to enhance the rapid microbiological diagnosis of HAP cases, and could aid in tailoring appropriate antibiotic therapies.

Tracking the history of circulating nucleic acids for cancer research in Brazil: A systematic review

Introduction: Circulating nucleic acids can be obtainedby minimally invasive procedures based on liquid biopsy,which has emerged as a promising area of investigation forscreening and monitoring cancer treatment. Currently, testsbased on circulating nucleic acid analysis, specifically cellfreeDNA (cfDNA), are commercially available for diagnosticand prognostic investigation of a number of neoplasms. Objective:To describe the research on circulating nucleic acidmarkers for cancer prospecting in Brazil, since this area hasadvanced rapidly in recent years. Methods: In this systematicreview, we surveyed Brazilian publications in cancer researchfocused on cfDNA and cfRNA present in different fluids. BothMEDLINE-PUBMED and EMBASE databases were inspectedusing terms such as “circulating nucleic acids”, “cancer”, and“Brazil”. Results: The search returned 326 articles, in which28 Brazilian translational studies were eligible. Differentmethodologies were reported for different types of cancer,in which cfDNA from plasma was the most investigatedbiological material. Molecular investigations included quantification,somatic mutation, RNA expression, genotyping,microsatellites, blood protein interaction, and methylation.Discrepancies in the regional distribution of the studies werealso observed. Conclusion: Studies on circulating nucleic acidmarkers have advanced significantly in the oncology field,but many others are needed to better address the clinicalpractice in Brazil.

Digital Nucleic Acid Signal Amplification Platform for Highly Sensitive DNA Mutation Analysis.

Digital nucleic acid analysis technology has shown great application potential due to its excellent performance. However, most current digital nucleic acid detection methods are based on PCR or other template amplification strategies. Here, we present an alternative analysis platform based on digital nucleic acid signal amplification in droplets termed dNASA. Using a bead-based controllable extension bridged cascade signal amplification reaction, we achieved an ultralow background, high efficiency, and highly specific nucleic acid signal amplification analysis. As a "proof of concept", we demonstrated the feasibility of the proposed dNASA platform in single-base DNA mutation analysis using artificially synthesized samples. This platform provides innovative ideas for the field of digital nucleic acid analysis.

Tunable Duplex Semiquantitative Detection of Nucleic Acids with a Visual Lateral Flow Immunoassay Readout.

Quantitative nucleic acid amplification testing (NAAT) is a key enabling technology for infectious disease management, especially in instances where viral load informs therapeutic decisions. Inadequate access to quantitative NAATs remains a challenge to the successful deployment of antiretroviral therapy (ART) regimens for patients with chronic hepatitis B virus (CHB) in low resourced settings (LRS). Current field-deployable NAATs are generally qualitative (yes/no) rather than quantitative in nature, making them ill-suited for viral load monitoring programs for CHB patients. Here, we report the development of a proof-of-concept molecular diagnostic test, the semiquantitative ligation and amplification (SQLA) assay, which achieves semiquantitative detection of input target DNA at two independently tunable detection thresholds with a simple visual readout. The SQLA assay utilizes a duplex competitive thermophilic helicase-dependent amplification (tHDA) chemistry and can be performed in under 1 h.

Thin Film Microextraction Enables Rapid Isolation and Recovery of DNA for Downstream Amplification Assays.

Nucleic acid analysis has been at the forefront of the COVID-19 global health crisis where millions of diagnostic tests have been used to determine disease status as well as sequencing techniques that monitor the evolving genome of SARS-CoV-2. In this study, we report the development of a sample preparation method that decreases the time required for DNA isolation while significantly increasing the sensitivity of downstream analysis. Functionalized planar supports are modified with a polymeric ionic liquid sorbent coating to form thin film microextraction (TFME) devices. The extraction devices are shown to have a high affinity for DNA while also exhibiting high reproducibility and reusability. Using quantitative polymerase chain reaction (qPCR) analysis, the TFME devices are shown to require low equilibration times while achieving higher preconcentration factors than solid-phase microextraction (SPME) by extracting larger masses of DNA. Rapid desorption kinetics enable higher DNA recoveries using desorption solutions that are less inhibitory to qPCR and loop-mediated isothermal amplification (LAMP). To demonstrate the advantageous features of the TFME platform, a customized leuco crystal violet LAMP assay is used for visual detection of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial oral fluid samples. When coupled to the TFME platform, 100% of LAMP reactions were positive for SARS-CoV-2 compared to 66.7% obtained by SPME for a clinically relevant concentration of 4.80 × 106 DNA copies/mL.

Application of droplet digital PCR method for DNA methylation-based age prediction from saliva.

The recent studies reported that DNA methylation markers show changes with age, and expected that the DNA methylation markers can be effectively used for estimation of age in forensic genetics. In this study, we applied droplet digital PCR (ddPCR) method to investigate the DNA methylation pattern in the CpG sites, and we constructed an age prediction model based on the ddPCR method. The ddPCR is capable of highly sensitive quantitation of nucleic acid and detection of sequence variations in gene by separating the sample into large number of partitions and clonally amplifying nucleic acids in each partition. We extracted DNA from saliva samples collected from several age groups. The DNA was bisulfite converted and subjected to ddPCR using specifically designed primers and probes. The methylation ratio of each sample was calculated and correlation between the methylation ratio and the chronological age was analyzed. In the results, methylated DNA ratio at the 4 CpG sites (cg14361627, cg14361627, cg08928145 and cg07547549) showed strong correlation with chronological age. Percent-methylation values at 4 CpG markers and chronological ages of the 76 individuals were analyzed by multiple regression analysis, and we constructed an age prediction model. We observed a strong correlation (Spearman's rho=0.922) between predicted and chronological ages of 76 individuals with a MAD from chronological age of 3.3years. Collectively, the result in this study showed the potential applicability of ddPCR to predict age from saliva.

Differential release of extracellular vesicle tRNA from oxidative stressed renal cells and ischemic kidneys

While urine-based liquid biopsy has expanded to the analyses of extracellular nucleic acids, the potential of transfer RNA (tRNA) encapsulated within extracellular vesicles has not been explored as a new class of urine biomarkers for kidney injury. Using rat kidney and mouse tubular cell injury models, we tested if extracellular vesicle-loaded tRNA and their m1A (N1-methyladenosine) modification reflect oxidative stress of kidney injury and determined the mechanism of tRNA packaging into extracellular vesicles. We determined a set of extracellular vesicle-loaded, isoaccepting tRNAs differentially released after ischemia–reperfusion injury and oxidative stress. Next, we found that m1A modification of extracellular vesicle tRNAs, despite an increase of the methylated tRNAs in intracellular vesicles, showed little or no change under oxidative stress. Mechanistically, oxidative stress decreases tRNA loading into intracellular vesicles while the tRNA-loaded vesicles are accumulated due to decreased release of the vesicles from the cell surface. Furthermore, Maf1-mediated transcriptional repression of the tRNAs decreases the cargo availability for extracellular vesicle release in response to oxidative stress. Taken together, our data support that release of extracellular vesicle tRNAs reflects oxidative stress of kidney tubules which might be useful to detect ischemic kidney injury and could lead to rebalance protein translation under oxidative stress.

Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment

BackgroundSurveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes.MethodsThe diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests.ResultsOf the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment.ConclusionMalaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes.

Observing Mesoscopic Nucleic Acid Capacitance Effect and Mismatch Impact via Graphene Transistors.

This work reports a molecular-scale capacitance effect of the double helical nucleic acid duplex structure for the first time. By quantitatively conducting large sample measurements of the electrostatic field effect using a type of high-accuracy graphene transistor biosensor, an unusual charge-transport behavior is observed in which the end-immobilized nucleic acid duplexes can store a part of ionization electrons like molecular capacitors, other than electric conductors. To elucidate this discovery, a cascaded capacitive network model is proposed as a novel equivalent circuit of nucleic acid duplexes, expanding the point-charge approximation model, by which the partial charge-transport observation is reasonably attributed to an electron-redistribution behavior within the capacitive network. Furthermore, it is experimentally confirmed that base-pair mismatches hinder the charge transport in double helical duplexes, and lead to directly identifiable alterations in electrostatic field effects. The bioelectronic principle of mismatch impact is also self-consistently explained by the newly proposed capacitive network model. The mesoscopic nucleic acid capacitance effect may enable a new kind of label-free nucleic acid analysis tool based on electronic transistor devices. The in situ and real-time nucleic acid detections for virus biomarkers, somatic mutations, and genome editing off-target may thus be predictable.

Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material

The search for early disease markers and the development of diagnostic systems has recently been expanding within genomics. Genomic deoxyribonucleic acid (DNA), cell-free DNA (cfDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. It has been shown that cfDNA that enters the bloodstream, in particular, as a result of apoptosis, necrosis, active tumor secretion and metastasis, is of great importance for studying molecular mechanisms of the pathological process and application in clinical practice. Circulating nucleic acid analysis can be used to monitor response to treatment, assess drug resistance, and quantify minimal residual disease. The review article reflects the following information about the biomaterial: source of cfDNA, methods of cfDNA isolation, storage and use for the diagnosis of certain diseases. Cell-free DNA can be present in biological fluids such as blood, urine, saliva, synovial and cerebrospinal fluid. In most cases, cfDNA is isolated from blood derivatives (serum and plasma), while it is most correct to use blood plasma for cfDNA isolation. Optimal and economically justifiable is the use of ethylenediaminetetra-acetic acid tubes for taking blood and obtaining plasma with subsequent cfDNA isolation. There is evidence that the optimal shelf life in an ethylenediaminetetra-acetic acid tube from the moment of blood sampling to subsequent isolation is a 2-hour interval. After centrifugation, cfDNA in plasma (or serum) can be stored for a long time at a temperature of -80O C. Storage at -20O C is undesirable, since DNA fragmentation increases.

Biofluids manipulation methods for liquid biopsy in minimally-invasive assays

The Liquid Biopsy (LB) is an opportunity for non-invasive diagnosis and prognosis of various diseases. To date, it isn't possible to consider that tissue biopsy can represent a pathology entirety. Then, body fluids are rich in a large number and variety of biomarkers and they can provide information about several diseases.•Recently, other biological fluids, easy to be collected are rising for their significant content of biomarkers and for the possibility to collect and manipulate them without the intervention of medical staff.•The management of biological fluids requires suitable storage methods. Temperature, storage time and physical stresses due to sample handling can lead to chemical and physical changes that may induce sample degradation and incorrect analysis.•The reliability of a diagnostic or screening test depends on its sensitivity and specificity. As the liquid biopsy is a 'snapshot' of a pathophysiological condition, it is crucial that its components do not degrade due to the improper handling of the body fluid.In this review, some handling methods of Saliva, Urine, Stool, Seminal Fluid, Tears and Sweat samples will be described, as well as protocols to facilitate the analysis of metabolites, nucleic acids, proteins and Extracellular Vesicles (EVs) from those unusual body fluids.

Fluorescence based miniaturized microfluidic and nanofluidic systems for biomedical applications.

Over the last two decades miniaturized microfluidic and nanofluidic systems with fluorescence setup emerged as a powerful technological platform for diverse biomedical applications. Bio-macromolecules such as nucleic acids and proteins are the core cellular components, their single molecule analysis allow us to understand biological processes, disease creation and progression, and development of novel treatment policies. Design and development of foolproof treatment methods requires rigorously analysis of nucleic acids and proteins such as length quantifications, sequence profiling, sequence mapping, analysis of conformational changes, analysis and recognition of epigenetic changes, and their interactions with other biomolecules. Miniaturized microfluidic and nanofluidic systems with fluorescence spectroscopy enable worldwide researchers to perform nucleic acids and proteins extractions and single molecule analysis from the trace amount of biological samples. In the present chapter we mostly highlighted over one decade applications of microfluidic and nanofluidic systems for single cell micro ribonucleic acid (miRNA) isolation and detection, deoxyribonucleic acid (DNA) mapping, DNA barcoding, identification of epigenetic mark on single DNA molecule, DNA-protein interactions study, protein sensing, protein sequencing, protein binding kinetics and many other applications. We also presented the recently reported microfluidic platform for the preparation of reproducible unisize aggregation induced emission (AIE) active nanomaterials and their biological applications.

Performance Comparison of GeneXpert MTB/RIF, Gene Chip Technology, and Modified Roche Culture Method in Detecting Mycobacterium tuberculosis and Drug Susceptibility in Sputum.

Our aim of this study was to observe and analyze the performance of the real-time fluorescence quantitative nucleic acid amplification detection of Mycobacterium tuberculosis/rifampicin resistance (GeneXpert MTB/RIF), gene chip technology, and modified Roche culture method in detecting MTB in sputum submitted for inspection and drug susceptibility. Patients with smear-negative suspected pulmonary TB (n = 120) in our hospital were enrolled in this study using a random number table, and sputum samples submitted for inspection were tested by the GeneXpert MTB/RIF, gene chip technology, and modified Roche culture method. With clinical diagnosis as the gold standard, the performance (mainly sensitivity and specificity) of the above three detection methods in the diagnosis of MTB was compared. Next, the drug susceptibility test (DST) was carried out on sputum samples, tested positive by the three methods. With the solid culture results as the evaluation criteria, the performance of the three detection methods in the diagnosis MTB and DST was compared. When compared with the modified Roche culture method, the GeneXpert MTB/RIF had the highest positive rate and a shorter overall test duration (P < 0.05). In contrast with the gene chip technology, the GeneXpert MTB/RIF exhibited higher sensitivity and negative predictive value (NPV) and lower specificity, accuracy, positive predictive value (PPV), and Kappa value (P < 0.05). According to analysis of the diagnostic performance of the three detection methods, GeneXpert MTB/RIF displayed the highest diagnostic sensitivity, ideal predictive values, and the highest similarity with clinical diagnosis in results (P < 0.05). The detection of susceptibility to isoniazid (INH) and RIF showed that the GeneXpert MTB/RIF and gene chip technology performed ideally in DST of MTB. In comparison with the modified Roche culture method, the GeneXpert MTB/RIF and gene chip technology have more prominent performance in detecting MTB and drug susceptibility. Besides, to further improve the accuracy of clinical diagnosis, various molecular biology detection methods can be combined to avoid delaying of the best time for the diagnosis and treatment of the disease.

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

Integrated Dual Nucleic Acid Analysis by Slip Valve-Assisted Fluidic Chip Combined with CRISPR/Cas12a Detection

Integrated Dual Nucleic Acid Analysis by Slip Valve-Assisted Fluidic Chip Combined with CRISPR/Cas12a Detection

Highly accelerated isothermal nucleic acid amplifications by butanol dehydration: simple, more efficient, and ultrasensitive.

Enzyme-free isothermal amplification reactions for nucleic acid analysis usually take several hours to obtain sufficient detection sensitivity, which limits their practical applications. Herein, we report a butanol dehydration-based method to greatly improve both the efficiency and the sensitivity of nucleic acid detections by three types of enzyme-free isothermal amplification reactions. The reaction time has been shortened from 3 h to 5-20 min with higher sensitivities. Especially in the DNAzyme-based amplification, the detection limit can be lowered over 16 000-fold to 3 × 10-17 mol L-1 in 2 h compared to the normal 3 h-reaction. We demonstrate that the high amplification efficiencies are attributed to the greatly accelerated reaction rates in the extremely concentrated reaction solutions caused by the butanol dehydration. This approach enhances the potential of applications of isothermal amplification reactions in clinical rapid tests, nanostructure synthesis, etc. and is promising to expand to other types of chemical reactions.

Optical Design of a Quantitative Microvolume Nucleic Acid Spectrophotometer with Non-Optical Fiber and All Radiation-Hardened Lens Elements

The purity of the nucleic acid samples obtained by extraction/precipitation or adsorption chromatography must be verified with microvolume spectrophotometry to ensure a high success rate of the subsequent nucleic acid sequencing while exploring the trace rare nucleic acids in space exploration with in-situ life detection. This paper reports an optical design for a radiation-hardened quantitative microvolume spectrophotometer with all radiation-hardened lens elements for space exploration instruments by using a non-optical fiber optical path with radiation-hardened optical glass elements. The results showed that the mean absolute error rate of the measured standard ribonucleic acid samples at concentrations between 50 ng/μL and 2300 ng/μL was within 2% when compared with a LINKO LKU–6000 ultraviolet–visible spectrophotometer.

Multianalyte lateral flow immunoassay for simultaneous detection of protein-based inflammation biomarkers and pathogen DNA

The detection of multiple biomolecule classes in one go is highly desirable for a wide variety of areas, and in particular for point-of-care diagnostics. For example, wound infections are a major problem for patient’s health and cause huge efforts in our healthcare system. In this regard, monitoring infected wounds through simultaneous detection of pathogens via nucleic acid analysis and detection of local inflammation biomarkers is key in order to enable a personalized therapy, improve the clinical outcome and thus, leading to a reduction of overall healthcare costs. In this regard, wound exudate offers an attractive sample material which can be collected in a non-invasive manner. Here, we report the development of a Multianalyte-Assay detecting inflammation biomarkers and pathogen DNA simultaneously from one sample within 35 min. Protein-compatible amplification and labeling transforms nucleic acid information into the measurement principle for protein detection. The combination with rapid detection via lateral flow immunoassay enables a fast and straightforward analysis of multiple biomolecule classes using identical assay conditions. To demonstrate the feasibility of the Multianalyte-Assay, the proinflammatory cytokine interleukin-6 (IL-6) and gDNA of the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) are used. The detection limits of 4 ng/mL IL-6 and 70 copies/reaction P. aeruginosa gDNA meet the clinically relevant range and thus, having tremendous potential to improve the wound management at the point-of-care.