Search Results (82)

Highly sensitive and non-invasive detection of macroorganisms using environmental nucleic acids (eNA) has transformed biosecurity surveillance. However, the persistence of legacy DNA compromises the spatiotemporal accuracy of environmental DNA (eDNA)-based detection, leading to false indications of contemporary species presence. This review critically evaluates emerging molecular approaches aimed at improving the temporal resolution of eNA signals and distinguishing living organisms from historical residues. We examine environmental RNA (eRNA), long-fragment eDNA (LFeDNA), propidium monoazide (PMA) treatment, and organelle-to-nuclear DNA ratios as indicators of eDNA age, assessing their principles, technical challenges, and practical potential. While eRNA offers the strongest theoretical link to organism viability, its application is constrained by rapid decay and stringent handling requirements. LFeDNA presents a more practical alternative but requires careful assay design. PMA treatment has shown limited effectiveness in excluding legacy DNA, whereas organelle-to-nuclear DNA ratios remain promising but underexplored. We identify key research priorities needed to transition these approaches from experimental studies to operational biosecurity tools. Addressing these gaps will improve interpretation of eNA signals, enabling more accurate detection of living invasive organisms and enhancing the reliability of biosecurity surveillance. Full article

West Nile virus (WNV) diagnosis relies on nucleic acid amplifications, but these techniques do not discriminate between infectious and non-infectious viral particles. This limitation can be bypassed by using a genome-binding dye (PMAxx) that is unable to cross membranes and can only bind to the genomes of non-intact (i.e., non-infectious) viral particles. This study evaluated a workflow combining PMAxx treatment with digital PCR to improve the molecular discrimination of intact WNV particles. Fifty-five samples (35 plasma, 20 urine) from 41 patients with WNV fever (WNF) or WNV neuroinvasive disease (WNND) were analyzed. Samples were tested with/without PMAxx treatment. Overall, PMAxx treatment resulted in a significant reduction in detectable viral RNA (median reduction: 1.0 Log copies/mL; p < 0.0001), indicating that a substantial fraction of RNA detected by standard methods originated from non-infectious particles. This reduction was more visible in urine (1.8 Log copies/mL) than in plasma (0.4 Log copies/mL), suggesting a higher proportion of degraded viral particles or free RNA in urine. Stratification by clinical presentation showed significant reductions in both WNF and WNND patients, with no significant differences between groups. This approach may represent a valuable adjunct for improving diagnostic interpretation and epidemiological assessment of WNV infection, particularly in matrices characterized by prolonged RNA persistence. Full article

Large wastewater treatment plants (WWTP) are increasingly supplemented with quaternary treatment. Classical monitoring hereby relies mostly on the measurement of oxygen demand, micropollutants and the nutrients phosphorus and nitrogen. From a microbiological perspective, relevant parameters to assess treatment performance include the removal efficacies of the fecal gene load as a proxy of pathogenic risk, antibiotic resistance genes and the bacterial regrowth potential. For this purpose, a combination of flow cytometry and quantitative PCR, together with a viability assessment, was applied to characterize a full-scale pilot plant. The pilot plant comprised conventional treatment and MBR and ozonation for advanced treatment. The assessment of fecal gene load was based on the quantification of Bacteroidales of human origin, as these obligate anaerobic bacteria cannot replicate within wastewater treatment plants. Whereas conventional treatment resulted in only moderate removal of these parameters, quaternary treatment typically led to a much stronger decrease. MBR treatment contributed most strongly to the removal with an appr. 6 log reduction compared to the primary clarification effluent, corroborating its microbiological merit for wastewater treatment. In addition to removing microorganisms and their genetic content, data also suggested a 95% reduction in extracellular DNA. Ozonation further enhanced microbiological removal. From an analytical perspective, the study shows the added value of using a long amplicon qPCR approach together with sample treatment with a viability dye to minimize false-positive signals and to avoid underestimation of treatment performance. The chosen diagnostic approach shows promise in assessing the microbiological treatment efficacy of WWTPs and as a basis to decide on the microbiological necessity of treatment upgrades. Full article

Rubber trees are crucial to the global industrial economy, but they are facing the threat of powdery mildew caused by Erysiphe quercicola. Effective management of this disease depends on early detection. However, traditional monitoring methods are labor-intensive and often inaccurate. This limitation underscores the need for more precise and efficient techniques. This study developed and validated an integrated molecular detection platform that combines quantitative PCR (qPCR), droplet digital PCR (ddPCR), and propidium monoazide (PMA) treatments. The platform demonstrated a robust detection range, accurately quantifying E. quercicola at concentrations as low as 10 spores/mL spore DNA and 10⁻⁵ ng/μL mycelial DNA. Additionally, the system distinguished viable from non-viable spores and detected E. quercicola mycelia in both asymptomatic leaves and aged lesions, significantly enhancing early-stage detection and disease monitoring. This technology also helps assess the efficacy of fungicides against powdery mildew, potentially reducing the use of chemicals and their environmental impact. By improving early diagnosis and disease management, this approach promises to reduce dependence on fungicides and mitigate economic and environmental impacts, highlighting the enormous potential of advanced molecular technologies in sustainable agricultural practices in rubber plantations. Full article

Wastewater-based epidemiology (WBE) is an effective tool for assessing health risks in rural areas with limited access to health care. Wastewater treatment plants (WWTPs) allow for the monitoring of pathogenic microorganisms, which is key to detecting viral integrity and bacterial viability to assess health risks. This study evaluated five rural WWTPs in Chile during 2022 in two seasons (autumn–winter and spring–summer). SARS-CoV-2, norovirus GI/GII, and HAdV-F40/41 was analyzed, along with antibiotic-resistant coliforms. Influent and effluent samples were used, with viral integrity analysis by propidium monoazide and culture methods to assess bacterial resistance. Despite the low number of clinical cases, SARS-CoV-2 was detected in all influent samples. Intact viral particles of NoV GI (78%), NoV GII (72%), and HAdV-F40/41 (65%) were found. This suggests that they may still be infectious. Viral removal ranged from 74% to 100%, although intact HAdV was detected in effluent (6.2%). Coliforms resistant to various antibiotics were detected and partially removed (22–100%). Removal efficiency depends on the type of treatment and the season of the year. WWTPs act as temporary reservoirs of infectious agents. This study reinforces the usefulness of WBE in rural contexts and WWTPs as barriers or not to these contaminants to the environment. Full article

Periodontitis is a biofilm-driven inflammatory disease in which conventional indices (probing depth, clinical attachment level, and radiographs) quantify tissue destruction without capturing the biology of infection. In this review, we synthesized microbiological diagnostics, from chairside tools to omics. We outline sampling strategies and emphasize the quantitative monitoring of bacterial load. Enzymatic assays (e.g., N-benzoyl-DL-arginine-2-naphthylamide hydrolysis assay test) measure functional activity at the point of care. Immunological methods include rapid immunochromatography for Porphyromonas gingivalis and enzyme-linked immunosorbent assay for the high-throughput measurement of bacterial antigens. Molecular platforms encompass quantitative polymerase chain reaction (qPCR) (TaqMan, SYBR, multiplex panels; propidium monoazide quantitative-qPCR for viable cells), checkerboard DNA–DNA hybridization for semi-quantitative community profiling, loop-mediated isothermal amplification (LAMP)/molecular beacon-LAMP for portable isothermal detection, and microarrays. Complementary modalities such as fluorescent in situ hybridization, next-generation sequencing, and Fourier transform infrared spectroscopy provide spatial, ecological, and biochemical resolutions. We discuss the limitations of current approaches, including sampling bias, presence–activity discordance, semi-quantitation, method biases, limited strain/function resolution, low-biomass artifacts, and lack of validated cutoffs. To address these challenges, we propose a pragmatic hybrid strategy: site-specific quantitative panels combined with activity and host-response markers interpreted alongside clinical metrics under standardized quality assurance/quality control. Priorities include outcome-linked thresholds, strain-aware/functional panels, robust point-of-care chemistry, and harmonized protocols to enable personalized periodontal care. Full article

Pseudomonas fluorescens is the main spoilage bacterium in milk, and its proliferation is one of the factors leading to the deterioration of the quality of raw milk. In this study, a rapid detection system for P. fluorescens was developed based on recombinase-aided amplification combined with a test strip (RAA-TS), which contained a double test line (DTL) targeting the virulence gene aprX of P. fluorescens and the housekeeping gene gyrB of Pseudomonas. Visual observation could detect gyrB (50 CFU/mL) and aprX (250 CFU/mL) within 90 min, including sample pretreatment and RAA reaction and detection steps. No cross-reactions were observed with Pseudomonas or other bacteria (n = 19). The quantitative detection limits (LOD) of gyrB and aprX for P. fluorescens in milk were 37 CFU/mL and 233 CFU/mL, respectively. Compared with polymerase chain reaction-agarose gel electrophoresis (PCR-AGE), the sensitivity of the developed RAA-TS-DTL system was increased by approximately four times. Furthermore, it could detect live P. fluorescens in milk when combined with optimized sample pretreatment by propidium monoazide (PMAxx). Its consistency with the traditional culture method in the detection of P. fluorescens spiked in milk samples (n = 25) was 100%. The developed RAA-TS-DTL had the advantages of high accuracy and short time consumption. Thus, it provides a new way or tool for the rapid screening or detection of P. fluorescens in milk. Full article

Torquetenovirus (TTV) is a ubiquitous, non-pathogenic DNA virus that has been suggested as a biomarker of immune competence, with the viral load correlating with the level of immunosuppression. However, by detecting non-intact viral particles, standard PCR-based quantification may overestimate the TTV viremia. To improve the clinical relevance of TTV quantification, in this study, we investigated the use of PMAxx™, a virion viability dye that selectively blocks the amplification of compromised virions. Serum samples from 10 Hepatitis C Virus-positive (HCV+) individuals, 81 liver transplant recipients (LTRs), and 40 people with HIV (PWH) were treated with PMAxx™ and analyzed for TTV DNA loads by digital droplet PCR (ddPCR). Furthermore, anti-SARS-CoV-2 IgG levels and neutralizing antibody (nAbs) titers were measured post-COVID-19 vaccination. Using ddPCR, the PMAxx™ treatment significantly reduced the TTV DNA levels in all the groups (mean reduction: 0.66 Log copies/mL), indicating the abundant presence of non-intact, circulating viral genomes. However, correlations between TTV DNA and SARS-CoV-2 IgG or nAbs were weak or absent in both PMAxx™-treated and untreated samples. These findings suggest that while PMAxx™ enhanced the specificity of TTV quantification, it did not improve the predictive value of TTV viremia at assessing vaccine-induced humoral responses. Full article

Urban sewage, aquaculture wastewater, and medical wastewater are significant reservoirs and transmission sources of Salmonella. Rapid detection of Salmonella is crucial for effectively reducing the risk of disease transmission and safeguarding public health. Differentiating viable Salmonella from inactivated cells presents significant challenges, affecting the accurate assessment of pathogen risks. Moreover, current detection methods face several limitations, including lengthy detection periods, high costs, and limited applicability, underscoring the need for rapid, sensitive, and visual detection diagnostic approaches. In this study, we combined propidium monoazide (PMA) with recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeats (CRISPR)/Cas12a systems to develop a rapid detection system for viable Salmonella targeting the fimY gene. DNA of viable Salmonella was amplified and visually detected within 60 min and dead cells were effectively excluded. We assessed the specificity and sensitivity of the PMA-RPA-CRISPR/Cas12a assay. The results showed that the assay had a high level of specificity, with no reactions observed with other pathogens. The application of PMA has no effect on the sensitivity of RPA-CRISPR/Cas12a technology and the visibility of the fluorescence reporting system. We successfully detected viable Salmonella in wastewater with a minimum detection limit of 10¹ CFU/mL. In summary, the PMA-RPA-CRISPR/Cas12a system developed in this study allows for the rapid and visual detection of viable Salmonella in wastewater at concentrations as low as 10¹ CFU/mL. By integrating PMA with the RPA-CRISPR/Cas12a technology, this system offers valuable technical support for the efficient, sensitive, and clear detection of viable Salmonella in wastewater. Full article

Bacteria are ubiquitous in the environment and critical to human health and disease, yet only a small fraction can be identified through standard culture methods. Advances in next-generation sequencing techniques have improved bacterial identification, but these DNA-based methods cannot distinguish live bacteria from relic DNA. Recently, DNA-labeling dyes (e.g., 5-bromo-2′-deoxyuridine [BrdU] and propidium monoazide [PMA]) have been used to detect metabolically active bacteria in different sample types. Here, we compare BrdU and PMA in combination with 16SrRNA gene sequencing to characterize metabolically active bacteria in two different sample types: (1) manufactured products (n = 78; cigarettes, hookah, and little cigar) and (2) natural samples (n = 186; rainwater, soil, and produce). Metabolically active bacterial communities identified in BrdU-labeled samples had lower alpha diversity than that of PMA-treated and non-treated samples. Pseudomonas, Sphingomonas, Enterobacter, and Acinetobacter were observed in all the samples tested. Irrespective of sample type, Pseudomonas was predominant in BrdU-treated samples, while Acinetobacter was more abundant in non-treated samples compared to PMA-treated samples. We also observed that PMA-treated samples tend to overestimate the metabolically active bacterial fraction compared to BrdU-treated samples. Overall, our study highlights how different labeling techniques influence bacterial community analysis findings, underscoring the need for careful selection of labeling approaches when assessing environmental samples. Full article

A requirement of any foodborne pathogen testing method is that it only detects live bacteria. Ethidium monoazide (EMA) and propidium monoazide (PMA) are dyes that penetrate the membranes of dead cells and form cross-linkages in the DNA, which prevents its amplification in PCR. This study investigated whether treatment with EMA or PMA would inhibit the sequencing of DNA from dead Escherichia coli. Range finding experiments with qPCR were conducted to determine the optimal concentrations of EMA and PMA needed to inhibit the amplification of DNA from dead cells while not influencing live cells. An EMA concentration that differentiated between live and dead cells could not be established. However, a PMA concentration of 25 µM effectively prevented qPCR amplification of DNA from dead E. coli while not impacting the amplification of live E. coli DNA. Sequencing experiments were conducted with PMA-treated live, untreated live, PMA-treated dead, and untreated dead E. coli. There were no significant differences in the detection of virulence genes of interest between the PMA-treated live, untreated live, and untreated dead E. coli. However, no DNA sequencing data were obtained from the PMA-treated dead E. coli. These results suggest that PMA could be incorporated into sample preparation methods prior to sequencing to selectively detect live cells of foodborne pathogens. Full article

Pseudomonas syringae pv. actinidiae (Psa) is responsible for causing kiwifruit canker disease. The detection of Psa is commonly carried out using normal PCR and culture-based isolation. However, normal PCR does not differentiate between live and dead cells, potentially resulting in the incorrect estimation of the amount of infectious substance in a sample. Such an incorrect estimation could result in unnecessary phytosanitary strategies and control measures. This study attempts to establish a specific assay for detecting only live Psa bacterial cells. To achieve this, a pair of strain-specific primers designed from HopZ3 effector were used, and the traditional PCR method was assessed using a nucleic acid-binding dye (propidium monoazide—PMA), establishing a PMA–PCR system and conditions for detecting live Psa in this study. Sensitivity tests showed a detection limit of 10 cfu/mL and 1 pg/μL. This method was also tested in diseased kiwifruit tissues and can be seen as a rapid and dependable replacement to PCR methods for detecting only those infective kiwifruit materials with viable Psa. Full article

Mpox, caused by the Monkeypox virus (MPV), is a global public health threat. Virus isolation is the gold standard to confirm MPV infection, but this process can face many challenges. As an alternative, a new method was developed in in vitro settings using 50 µM of propidium monoazide (PMAxx, a DNA-binding agent) coupled with digital droplet PCR (ddPCR). Frozen clinical samples analyzed by PMAxx-ddPCR had a median of 0.8 copies/µL, while untreated samples had a median of 29.8 copies/µL. Since a substantial percentage of reduction was observed in these samples (>80%), it was verified whether this reduction could be due to the freezing process. This hypothesis was confirmed both in vitro and using clinical samples. A gradual increase in the mean percentage of reduction was observed after freezing–thawing cycles of MPV-isolate (59.5−81.4%). Moreover, a different percentage of reduction was observed before (68.2%) and after freezing (97.4%) the specimens, suggesting that the freezing process could reduce the number of complete viral particles. Our study shows strong evidence of the usefulness of PMAxx in clinical settings. PMAxx ensures the detection of intact MPV particles, which improves the accuracy of MPV load measurements. This method not only increases the reliability of MPV diagnosis but also overcomes virus isolation limitations. Full article

Accurate detection of viable Escherichia coli O157:H7 in fresh produce wash water is critical for ensuring food safety and mitigating foodborne illnesses. This study evaluated an optimized on-filter propidium monoazide (PMA)-quantitative PCR (qPCR) method for detecting viable E. coli O157:H7 in romaine lettuce wash water, involving PMA pretreatment on a filter to block DNA amplification from dead cells. The method consistently detected viable cells across chemical oxygen demand (COD) levels of 1000 and 200 mg O₂/L, with no significant differences (p > 0.05), indicating its tolerance to organic matter interference. Optimization experiments identified 10 µM PMA with a 10 min exposure time as the most effective pretreatment, achieving efficient inhibition of DNA from dead cells while preserving viable cell integrity. The limit of detection (LOD) was 1.3 CFU/mL, confirming its suitability for detecting low bacterial loads. Performance evaluations revealed that PMA-qPCR was accurate at viable-to-dead cell ratios of 1:10 or higher but became less reliable when dead cells outnumbered viable cells by a factor of 10 or more. The study demonstrates the potential of on-filter PMA-qPCR for routine food safety monitoring protocols in the fresh produce industry, while highlighting the critical role of viable-to-dead cell ratios in ensuring accurate detection, particularly in challenging samples with high dead cell loads. Full article

In this study, the performance of quantitative PCR, combined or not with propidium monoazide (PMA), to recover Salmonella from peanut products after different storage times was evaluated. The samples were inoculated with 5–6 log cfu g⁻¹ of Salmonella Typhimurium ATCC 14028 and stored at 28 °C for up to 540 d. The correlation between the threshold cycle number (Ct) and the colony-forming units (cfu) was obtained by a standard curve, which showed a linear correlation (R² = 0.97). The highest counts were recovered by qPCR (p < 0.05); however, it quantified both viable and non-viable cells. For roasted peanuts, a significant difference (p < 0.05) between qPCR-PMA and the culture method was verified only for samples stored for 30 d, i.e., 2.8 versus 4.0 log cfu g⁻¹. Further, there was no VBNC status in the roasted peanuts, even after long-term exposure to desiccation stress. For peanut-based products, after 540 d, only paçoca showed a significant difference (p < 0.05) among the three methods evaluated. In peanut brittle, qPCR-PMA detected 1.5 log cfu g⁻¹, while, in the culture method, Salmonella was recovered in 1 g. The pathogen was below the detection limit in pé-de-moça either by plate count or qPCR-PMA. Therefore, qPCR-PMA shows potential for use in quantifying Salmonella in peanut products. Full article