Search Results (59)

Accurate gene expression quantification using reverse transcription quantitative PCR (RT-qPCR) requires stable reference genes (RGs) for reliable normalization. However, few studies have systematically identified RGs suitable for simultaneous high salt, alkaline, and high-temperature conditions. This study addresses this gap by evaluating the stability of eight candidate RGs in the anaerobic halophilic alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF^T under combined salt, alkali, and thermal stresses. The stability of these candidate RGs was assessed using five statistical algorithms: Delta CT, geNorm, NormFinder, BestKeeper, and RefFinder. Results indicated that recA exhibited the highest expression stability across all tested conditions and proved adequate as a single RG for normalization in both RT-qPCR and droplet digital PCR (ddPCR) assays. Furthermore, recA alone or combined with other RGs (sigA, rsmH) effectively normalized the expression of seven stress-response genes (proX, opuAC, mnhE, nhaC, trkH, ducA, and pimT). This work represents the first systematic validation of RGs under polyextreme stress conditions, providing essential guidelines for future gene expression studies in extreme environments and aiding research on microbial adaptation mechanisms in halophilic, alkaliphilic, and thermophilic microorganisms. Full article

Background/Objectives: Cxbladder^® Triage Plus is a multimodal urinary biomarker assay that combines reverse transcription-quantitative analysis of five mRNA targets and droplet-digital polymerase chain reaction (ddPCR) analysis of six DNA single-nucleotide variants (SNVs) from two genes (fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT)) to provide risk stratification for urothelial carcinoma (UC) in patients with hematuria. This study evaluated the analytical validity of Triage Plus. Methods: The development dataset used urine samples from patients with microhematuria or gross hematuria that were previously stabilized with Cxbladder solution. Triage Plus was evaluated for predicted performance, analytical criteria (linearity, sensitivity, specificity, accuracy, and precision), extraction efficiency, and inter-laboratory reproducibility. Results: The development dataset included 987 hematuria samples. Compared with cystoscopy (standard of care), Triage Plus had a predicted sensitivity of 93.6%, specificity of 90.8%, positive predictive value (PPV) of 46.5%, negative predictive value of 99.4%, and test-negative rate of 84.1% (score threshold 0.15); the PPV increased to 74.6% for the 0.54 score threshold. For the individual FGFR3 and TERT SNVs, the limit of detection (analytical sensitivity) was a mutant-to-wild type DNA ratio of 1:440–1:1250 copies/mL. Intra- and inter-assay variance was low, while extraction efficiency was high. All other pre-specified analytical criteria (linearity, specificity, and accuracy) were met. Triage Plus showed good reproducibility (87.9% concordance between laboratories). Conclusions: Cxbladder Triage Plus accurately and reproducibly detected FGFR3 and TERT SNVs and, in combination with mRNA expression, provides a non-invasive, highly sensitive, and reproducible tool that aids in risk stratification of patients with hematuria. Full article

Toxoplasma gondii, Giardia intestinalis, and Cryptosporidium spp. are common pathogens that contaminate water and food. They can pose serious health risks, especially to vulnerable groups like immunocompromised individuals, pregnant women, young children, and aging people. An all-encompassing approach to minimizing transmission involves identifying effective techniques for detecting, treating, and preventing protozoan parasites. This study confirmed the effectiveness of a Droplet Digital Reverse Transcription Polymerase Chain Reaction (dd RT-PCR) method for quickly and accurately identifying Toxoplasma gondii, Giardia intestinalis, and Cryptosporidium species in honeybees, honey, and pollen by using ISO 17468 and ISO 16140 standard guidelines. The study evaluated honeybee (n = 16), honey (n = 12), and pollen (n = 8) samples collected from various apiaries in Southern Italy between June and September 2023. The results showed that honeybees, honey, and pollen can be considered bioindicators of infections by T. gondii, G. intestinalis, and Cryptosporidium spp. Furthermore, pollen, along with honey to a lesser degree, can serve as significant indicators for evaluating food safety. Therefore, it is essential to monitor their quality and purity due to environmental influences. Full article

Clinical studies indicate the etiology of liver disease to be unknown in 5% to 30% of patients. A long-standing hypothesis is the existence of unknown viruses beyond hepatitis A through E virus. We conducted serum virome sequencing in nine patients with cryptogenic liver disease and identified eight contigs that could not be annotated. One was determined to be a contaminant, while two of seven contigs from an individual (Patient 3) were validated by reverse transcription and polymerase chain reaction (RT-PCR) and Sanger sequencing. The possibility of contamination was completely excluded through PCR, with templates extracted using different methods from samples taken at different time points. One of the contigs, Seq260, was characterized as negative-sense single-stranded DNA via enzymatic digestion and genome walking. Digital-droplet PCR revealed the copy number of Seq260 to be low: 343 copies/mL. Seq260-based nested PCR screening was negative in 200 blood donors and 225 patients with liver disease with/without known etiologies. None of the seven contigs from Patient 3 was mapped onto 118,713 viral metagenomic data. Conclusively, we discovered seven unknown contigs from a patient with cryptogenic liver cirrhosis. These sequences are likely from a novel human virus with a negative-sense, linear single-stranded DNA genome. Full article

The aim of this study is to ascertain whether qRT-PCR (reverse transcriptase real-time PCR) or RT-ddPCR (reverse transcriptase digital droplet PCR) is more effective for detecting SARS-CoV-2 RNA (severe acute respiratory syndrome coronavirus 2 RNA) in blood plasma from COVID-19 (coronavirus infectious disease-19) patients. The E-gene of SARS-CoV-2 RNA was quantified using both methods in 128 plasma samples from 70 hospitalized patients, followed by a statistical analysis to compare the sensitivity and concordance between the methods. Out of the 128 samples, 89 yielded consistent results irrespective of the method used, whereas 39 samples showed discrepancies between the two different methods. RT-ddPCR frequently registered higher viral quantities compared to qRT-PCR; however, the results did not demonstrate a clear superiority in sensitivity for RT-ddPCR. Although RT-ddPCR registered higher viral quantities, this study concludes that both methods provide comparable results for detecting SARS-CoV-2 E-gene RNA in plasma. Full article

Respiratory viral infections trigger immune and inflammatory responses that can be associated with excessive oxidative stress, glutathione (GSH) depletion, and a cytokine storm that drives virus-induced cell/tissue damage and severe disease. Erdosteine is a thiol-based drug with proven mucolytic, anti-inflammatory, antioxidant, and antibacterial properties, but less is known about its antiviral effects. We performed in vitro studies to investigate the antiviral and anti-inflammatory activity of erdosteine in A549-hACE2 human lung epithelial cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or respiratory syncytial virus (RSV) and in Caco-2 human colon carcinoma cells infected with influenza A virus (H1N1). The cells were treated with different concentrations of erdosteine or its active metabolite 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MET-1) before and after viral infection. The viral replication/load in the cell culture supernatants was measured by real-time quantitative polymerase chain reaction (RT-qPCR) assay and digital droplet PCR. The gene expression of innate immune response signaling pathways and oxidative stress was analyzed by reverse transcription PCR custom-array. The results showed that erdosteine and its active metabolite, at concentrations consistent with an approved therapeutic human dosage, were not directly cytotoxic and had significant antiviral effects in cells pre-infected with SARS-CoV-2, RSV, and H1N1. The transcriptome analysis showed that erdosteine activated innate immune responses by stimulating overexpression of type I interferon and inflammasome pathways and modulated oxidative stress by inducing the modulation of oxidative stress and GSH pathways. These findings suggest that erdosteine may be a useful treatment for respiratory viral infections. Full article

Foodborne viruses are significant contributors to global food safety incidents, posing a serious burden on human health and food safety. In this study, a multiplex reverse transcription–droplet digital PCR (RT-ddPCR) assay based on the MS2 phage as a process control virus (PCV) was developed to achieve the simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in bivalve shellfish. By optimizing the reaction system and procedures, the best reaction conditions were selected, and the specificity, sensitivity, and reproducibility of the method were assessed. Additionally, the MS2 phage’s recovery rate was utilized as an indicator to evaluate the optimal sample nucleic acid enrichment method. The results indicated that the RT-ddPCR assay exhibited optimal amplification efficiency with primer concentrations of 900 nmol/L, probe concentrations of 350 nmol/L for HAV and HEV, and 500 nmol/L for MS2, an annealing temperature of 53.1 °C, an extension time of 90 s, and 45 cycles. Additionally, the developed multiplex RT-ddPCR assay demonstrated high specificity, with quantitation limits of 12.6, 8.9, and 7.8 copies/reaction being observed for HAV, HEV, and the MS2 phage, respectively. A total of 240 bivalve samples were analyzed, of which 4 were positive for HAV and 12 for HEV. The viral loads for HAV ranged from 3048 to 6528 copies/2 g, while those for HEV ranged from 3312 to 20,350 copies/2 g. This assay provides a vital tool for enhancing food safety monitoring. Full article

Plum pox virus (PPV) is the etiological agent of sharka, the most important viral disease of stone fruit worldwide. In this study, a one-step reverse transcription real-time PCR test (RT-qPCR) was modified and translated as a one-step RT-droplet digital PCR (RT-ddPCR) for sensitive, direct, and accurate detection and quantification of PPV. The modified RT-qPCR and RT-ddPCR PPV detection tests were validated using both plant purified total RNA (TRNA) and crude extract as templates. The proposed tests were sensitive, specific, selective, repeatable, and reproducible in detecting PPV from fresh, lyophilized, and in vitro plant samples. RT-ddPCR was more sensitive than RT-qPCR in detecting PPV using purified TRNA while showing the same sensitivity using crude extract. This work highlights the robustness, time-saving, and cost-effective nature of the proposed one-step RT-ddPCR test, offering a potential reduction in resources for PPV detection and quantification even with raw extracts. Full article

Canine distemper virus (CDV) poses a substantial threat to diverse carnivorans, leading to systemic and often fatal diseases. Accurate and prompt diagnosis is paramount for effective management and curbing further transmission. This study evaluates the diagnostic performance of droplet digital PCR (ddPCR) in comparison to conventional reverse-transcription (RT-PCR) and quantitative reverse-transcription real-time PCR (RT-qPCR). Seventy-six clinical samples were collected from dogs with CDV symptoms diagnosed by specialized veterinarians, and sixteen samples from apparently healthy individuals. Conventional PCR, quantitative real-time PCR, and ddPCR were deployed, and their diagnostic capabilities were meticulously assessed. DdPCR exhibited heightened analytical sensitivity, reaching a detection limit of 3 copies/μL, whereas RT-qPCR had a detection limit of 86 copies/μL. The comparative analysis between clinical diagnosis and molecular techniques, including RT-PCR and RT-qPCR, demonstrated low concordance, with Kappa coefficients of 0.268 and 0.324, respectively. In contrast, ddPCR showed a moderate concordance, with a Kappa coefficient of 0.477. The sensitivity was 42.4% for RT-PCR, 57.9% for RT-qPCR, and 72.4% for ddPCR, with 100% specificity for all methods. This study underscores ddPCR’s superior sensitivity and agreement with clinical CDV diagnosis, even at low viral concentrations, suggesting it as a promising alternative for CDV diagnosis. Full article

Pine wilt disease (PWD), caused by the nematode Bursaphelenchus xylophilus, is a highly destructive forest disease that necessitates rapid and precise identification for effective management and control. This study evaluates various detection methods for PWD, including morphological diagnosis, molecular techniques, and remote sensing. While traditional methods are economical, they are limited by their inability to detect subtle or early changes and require considerable time and expertise. To overcome these challenges, this study emphasizes advanced molecular approaches such as real-time polymerase chain reaction (RT-PCR), droplet digital PCR (ddPCR), and loop-mediated isothermal amplification (LAMP) coupled with CRISPR/Cas12a, which offer fast and accurate pathogen detection. Additionally, DNA barcoding and microarrays facilitate species identification, and proteomics can provide insights into infection-specific protein signatures. The study also highlights remote sensing technologies, including satellite imagery and unmanned aerial vehicle (UAV)-based hyperspectral analysis, for their capability to monitor PWD by detecting asymptomatic diseases through changes in the spectral signatures of trees. Future research should focus on combining traditional and innovative techniques, refining visual inspection processes, developing rapid and portable diagnostic tools for field application, and exploring the potential of volatile organic compound analysis and machine learning algorithms for early disease detection. Integrating diverse methods and adopting innovative technologies are crucial to effectively control this lethal forest disease. Full article

Liquid biopsy has emerged as a promising noninvasive approach for colorectal cancer (CRC) management. This review focuses on technologies detecting circulating nucleic acids, specifically circulating tumor DNA (ctDNA) and circulating RNA (cfRNA), as CRC biomarkers. Recent advancements in molecular technologies have enabled sensitive and specific detection of tumor-derived genetic material in bodily fluids. These include quantitative real-time PCR, digital PCR, next-generation sequencing (NGS), and emerging nanotechnology-based methods. For ctDNA analysis, techniques such as BEAMing and droplet digital PCR offer high sensitivity in detecting rare mutant alleles, while NGS approaches provide comprehensive genomic profiling. cfRNA detection primarily utilizes qRT-PCR arrays, microarray platforms, and RNA sequencing for profiling circulating microRNAs and discovering novel RNA biomarkers. These technologies show potential in early CRC detection, treatment response monitoring, minimal residual disease assessment, and tumor evolution tracking. However, challenges remain in standardizing procedures, optimizing detection limits, and establishing clinical utility across disease stages. This review summarizes current circulating nucleic acid detection technologies, their CRC applications, and discusses future directions for clinical implementation. Full article

The ongoing COVID-19 pandemic, caused by the rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since early 2020, has highlighted the need for sensitive and reliable diagnostic methods. Droplet digital PCR (ddPCR) has demonstrated superior performance over the gold-standard reverse transcription PCR (RT-PCR) in detecting SARS-CoV-2. In this study, we explored the development of a multiplex ddPCR assay that enables sensitive quantification of SARS-CoV-2, which could be utilized for antiviral screening and the monitoring of COVID-19 patients. We designed a quadruplex ddPCR assay targeting four SARS-CoV-2 genes and evaluated its performance in terms of specificity, sensitivity, linearity, reproducibility, and precision using a two-color ddPCR detection system. The results showed that the quadruplex assay had comparable limits of detection and accuracy to the simplex ddPCR assays. Importantly, the quadruplex assay demonstrated significantly improved performance for samples with low viral loads and ambiguous results compared to the standard qRT-PCR approach. The developed multiplex ddPCR represents a valuable alternative and complementary tool for the diagnosis of SARS-CoV-2 and potentially other pathogens in various application scenarios beyond the current COVID-19 pandemic. The improved sensitivity and reliability of this assay could contribute to more effective disease monitoring and antiviral screening during the ongoing public health crisis. Full article

Enterovirus-D68 (EV-D68) is a reemerging virus that has been associated with numerous outbreaks in children in the past 10 years. Most assays examining viral infection kinetics have relied on the use of quantitative RT-PCR (qRT-PCR) assays as an assay of choice. Though valuable, there are inherent limitations that introduce variability, thereby reducing its value when comparing results across the field. Unlike the qRT-PCR assay that uses a standard curve to determine the copy number of viral RNA, the droplet digital PCR assay (ddPCR) directly quantifies the absolute number of copies within a given sample, which in turn makes the assay highly sensitive and accurate. Here, we have developed an EV-D68-specific ddPCR assay that effectively quantifies EV-D68 RNA copies in both cells and supernatants within a dynamic range of 6.7 × 10⁻³ copies/μL to 1.2 × 10⁴ copies/μL of the sample. The assay was highly specific for a broad range of EV-D68 isolates (Fermon, US/MO/14-18947, US/MO/14-18949, US/KY/14-18953, USA/2018-23088, USA/2020-23336 and EV-D68-infected human nasal turbinate samples from the 2022 outbreak) without cross-reactivity to other viruses such as Enterovirus-A71 (EV-A71), Human Parechovirus (HPeV)-1 and -2, Coxsackievirus (CV)-B1, Human Coronavirus (HCoV)-NL63, SARS-CoV-2, Influenza-A and B, Rhinovirus, and Respiratory Syncytial Virus (RSV)-A2, which are known to cause infection in children. The assay was able to readily quantify EV-D68 in infected cells and supernatants along with nasal turbinate samples collected from children during the 2022 outbreak. Our results suggest that the assay can be readily translated to accurately quantify viral loads in tissues and body fluids such as plasma and lung or nasal aspirates. Full article

Coronaviruses (CoVs), a subfamily of Orthocoronavirinae, are viruses that sometimes present a zoonotic character. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the recent outbreak of COVID-19, which, since its outbreak in 2019, has caused about 774,593,066 confirmed cases and 7,028,881 deaths. Aereosols are the main route of transmission among people; however, viral droplets can contaminate surfaces and fomites as well as particulate matter (PM) in suspensions of natural and human origin. Honey bees are well known bioindicators of the presence of pollutants and PMs in the environment as they can collect a great variety of substances during their foraging activities. The aim of this study was to evaluate the possible role of honey bees as bioindicators of the prevalence SARS-CoV-2. In this regard, 91 samples of honey bees and 6 of honey were collected from different apiaries of Campania region (Southern Italy) in four time periods from September 2020 to June 2022 and were analyzed with Droplet Digital RT-PCR for SARS-CoV-2 target genes Orf1b and N. The screening revealed the presence of SARS-CoV-2 in 12/91 in honey bee samples and in 2/6 honey samples. These results suggest that honey bees could also be used as indicators of outbreaks of airborne pathogens such as SARS-CoV-2. Full article

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC. Full article