Search Results (24)

Infectious laryngotracheitis virus (ILTV), a DNA virus classified as Gallid alphaherpesvirus 1, causes a highly contagious respiratory disease in chickens, leading to significant economic losses and health risks for the poultry industry. The rapid detection of ILTV is essential to control its spread and prevent outbreaks. Traditional diagnostic methods like PCR are costly, require specialized personnel, and delay response efforts. To address this, we developed a point-of-care diagnostic test combining loop-mediated isothermal amplification (LAMP) with DNA nanoprobes on respiratory swabs. LAMP targets the ILTV-glycoprotein E (gE) gene, enabling rapid nucleic acid amplification at 65 °C without extraction, making it suitable for on-site detection. DNA nanoprobes provide a colorimetric readout visible to the naked eye. Gold nanoparticles drive this readout, as their red color, based on localized surface plasmon resonance, persists in the presence of ILTV DNA through DNA-DNA hybridization, ensuring reliable detection. The assay achieved 100% sensitivity and specificity for ILTV-gE, with a detection limit of 200 copies per reaction, allowing for the early identification of infections. The results are available within 45 min, enabling prompt measures to control ILTV spread. Cost-effective and user-friendly, this method enhances disease management and biosecurity in poultry farms. Full article

Background: Tube thoracostomy, utilized through conventional methodologies in the context of pleural disorders such as pleural effusion and pneumothorax, constitutes one of the primary therapeutic interventions. Nonetheless, it is imperative to recognize that invasive procedures, including tube thoracostomy, are classified as aerosol-generating activities during the management of pleural conditions in patients afflicted with COVID-19, thus raising substantial concerns regarding the potential exposure of healthcare personnel to the virus. The objective of this investigation was to assess the SARS-CoV-2 viral load by detecting viral RNA in pleural drainage specimens from patients who underwent tube thoracostomy due to either pleural effusion or pneumothorax. Methods: In this single-center prospective cross-sectional analysis, a real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was employed to conduct swab tests for the qualitative identification of nucleic acid from SARS-CoV-2 in pleural fluids acquired during tube thoracostomy between August 2021 and December 2021. Results: All pleural drainage specimens from 21 patients who tested positive for COVID-19 via nasopharyngeal PCR, of which 14 underwent tube thoracostomy due to pneumothorax, 4 due to both pneumothorax and pleural effusion, and 3 due to pleural effusion, were found to be negative for SARS-CoV-2 RNA. Moreover, individuals exhibiting pleural effusion were admitted to the intensive care unit with a notably higher incidence, yet demonstrated significantly more radiological anomalies in patients diagnosed with pneumothorax. Conclusions: The current findings, inclusive of the results from this study, do not furnish scientific evidence to support the notion that SARS-CoV-2 is transmitted via aerosolization during tube thoracostomy, and it remains uncertain whether the virus can be adequately contained within pleural fluids. Full article

Human Papillomavirus (HPV) infection is a significant global health concern linked to various cancers, particularly cervical cancer. Timely and accurate detection of HPV is crucial for effective management and prevention strategies. Traditional laboratory-based HPV testing methods often suffer from limitations such as long turnaround times, restricted accessibility, and the need for trained personnel, especially in resource-limited settings. Consequently, there is a growing demand for point-of-care (POC) HPV testing solutions that offer rapid, easy-to-use, and convenient screening at the primary care level. This review provides a comprehensive overview of recent advancements and emerging technologies utilized in POC HPV testing using isothermal amplification methods, in addition to evaluating their diagnostic performance, sensitivity, specificity, and clinical utility compared to conventional laboratory-based assays, particularly in low-resource settings, where access to centralized laboratory facilities is limited. We provide insights into the potential of isothermal nucleic acid amplification to revolutionize cervical cancer screening and prevention efforts worldwide, with emphasis on the need for continued research, innovation, and collaboration to optimize the performance, accessibility, and affordability of POC HPV testing solutions, ultimately contributing to the worldwide effort towards the elimination of this disease. Full article

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness. Full article

Accurate and timely molecular diagnosis of respiratory diseases in chickens is essential for implementing effective control measures, preventing the spread of diseases within poultry flocks, minimizing economic loss, and guarding food security. Traditional molecular diagnostic methods like polymerase chain reaction (PCR) require expensive equipment and trained personnel, limiting their use to centralized labs with a significant delay between sample collection and results. Loop-mediated isothermal amplification (LAMP) of nucleic acids offers an attractive alternative for detecting respiratory viruses in broiler chickens with sensitivity comparable to that of PCR. LAMP’s main advantages over PCR are its constant incubation temperature (∼65 °C), high amplification efficiency, and contaminant tolerance, which reduce equipment complexity, cost, and power consumption and enable instrument-free tests. This review highlights effective LAMP methods and variants that have been developed for detecting respiratory viruses in chickens at the point of need. Full article

The human papillomavirus (HPV) is a non-enveloped DNA virus transmitted through skin-to-skin contact that infects epithelial and mucosal tissue. It has over 200 known genotypes, classified by their pathogenicity as high-risk and low-risk categories. High-risk HPV genotypes are associated with the development of different types of cancers, including cervical cancer, which is a leading cause of mortality in women. In clinical practice and the market, the principal tests used to detect HPV are based on cytology, hybrid detection, and qPCR. However, these methodologies may not be ideal for the required timely diagnosis. Tests have been developed based on isothermal nucleic acid amplification tests (INAATs) as alternatives. These tests offer multiple advantages over the qPCR, such as not requiring specialized laboratories, highly trained personnel, or expensive equipment like thermocyclers. This review analyzes the different INAATs applied for the detection of HPV, considering the specific characteristics of each test, including the HPV genotypes, gene target, the limit of detection (LOD), detection methods, and detection time. Additionally, we discuss the tests available on the market that are approved by the Food and Drug Administration (FDA). Finally, we address the challenges and potential solutions for the large-scale implementation of INAATs, particularly in rural or underserved areas. Full article

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article. Full article

We evaluated an active participatory design for the regional surveillance of notifiable swine pathogens based on testing 10 samples collected by farm personnel in each participating farm. To evaluate the performance of the design, public domain software was used to simulate the introduction and spread of a pathogen among 17,521 farms in a geographic region of 1,615,246 km². Using the simulated pathogen spread data, the probability of detecting ≥ 1 positive farms in the region was estimated as a function of the percent of participating farms (20%, 40%, 60%, 80%, 100%), farm-level detection probability (10%, 20%, 30%, 40%, 50%), and regional farm-level prevalence. At 0.1% prevalence (18 positive farms among 17,521 farms) and a farm-level detection probability of 30%, the participatory surveillance design achieved 67%, 90%, and 97% probability of detecting ≥ 1 positive farms in the region when producer participation was 20%, 40%, and 60%, respectively. The cost analysis assumed that 10 individual pig samples per farm would be pooled into 2 samples (5 pigs each) for testing. Depending on the specimen collected (serum or swab sample) and test format (nucleic acid or antibody detection), the cost per round of sampling ranged from EUR 0.017 to EUR 0.032 (USD 0.017 to USD 0.034) per pig in the region. Thus, the analysis suggested that an active regional participatory surveillance design could achieve detection at low prevalence and at a sustainable cost. Full article

Aims: To date, precision medicine has played a pivotal role in the clinical administration of solid-tumor patients. In this scenario, a rapidly increasing number of predictive biomarkers have been approved in diagnostic practice or are currently being investigated in clinical trials. A pitfall in molecular testing is the diagnostic routine sample available to analyze predictive biomarkers; a scant tissue sample often represents the only diagnostical source of nucleic acids with which to conduct molecular analysis. At the sight of these critical issues, next-generation sequencing (NGS) platforms emerged as referral testing strategies for the molecular analysis of predictive biomarkers in routine practice, but the need for highly skilled personnel and extensive working time drastically impacts the widespread diffusion of this technology in diagnostic settings. Here, we technically validate a fully integrated NGS platform on diagnostic routine tissue samples previously tested with an NGS-based diagnostic workflow by a referral institution. Methods: A retrospective series of n = 64 samples (n = 32 DNA, n = 32 RNA samples), previously tested using a customized NGS assay (SiRe™ and SiRe fusion), was retrieved from the internal archive of the University of Naples Federico II. Each sample was tested by adopting an Oncomine Precision Assay (OPA), which is able to detect 2769 molecular actionable alterations [hotspot mutations, copy number variations (CNV) and gene fusions] on fully integrated NGS platforms (Genexus, Thermo Fisher Scientific (Waltham, MA, USA). The concordance rate between these technical approaches was determined. Results: The Genexus system successfully carried out molecular analysis in all instances. A concordance rate of 96.9% (31 out of 32) was observed between the OPA and SiRe™ panels both for DNA- and RNA-based analysis. A negative predictive value of 100% and a positive predictive value of 96.9% (62 out of 64) were assessed. Conclusions: A fully automatized Genexus system combined with OPA (Thermo Fisher Scientific) may be considered a technically valuable, time-saving sequencing platform to test predictive biomarkers in diagnostic routine practice. Full article

Pathogenic pathogens invade the human body through various pathways, causing damage to host cells, tissues, and their functions, ultimately leading to the development of diseases and posing a threat to human health. The rapid and accurate detection of pathogenic pathogens in humans is crucial and pressing. Nucleic acid detection offers advantages such as higher sensitivity, accuracy, and specificity compared to antibody and antigen detection methods. However, conventional nucleic acid testing is time-consuming, labor-intensive, and requires sophisticated equipment and specialized medical personnel. Therefore, this review focuses on advanced nucleic acid testing systems that aim to address the issues of testing time, portability, degree of automation, and cross-contamination. These systems include extraction-free rapid nucleic acid testing, fully automated extraction, amplification, and detection, as well as fully enclosed testing and commercial nucleic acid testing equipment. Additionally, the biochemical methods used for extraction, amplification, and detection in nucleic acid testing are briefly described. We hope that this review will inspire further research and the development of more suitable extraction-free reagents and fully automated testing devices for rapid, point-of-care diagnostics. Full article

The development and optimization of lipid nanoparticle (LNP) formulations through hydrodynamic mixing is critical for ensuring the efficient and cost-effective supply of vaccines. Continuous LNP formation through microfluidic mixing can overcome manufacturing bottlenecks and enable the production of nucleic acid vaccines and therapeutics. Predictive process models developed within a QbD Biopharma 4.0 approach can ensure the quality and consistency of the manufacturing process. This study highlights the importance of continuous LNP formation through microfluidic mixing in ensuring high-quality, in-specification production. Both empty and nucleic acid-loaded LNPs are characterized, followed by a TFF/buffer exchange to obtain process parameters for the envisioned continuous SPTFF. It is shown that LNP generation by pipetting leads to a less preferable product when compared to continuous mixing due to the heterogeneity and large particle size of the resulting LNPs (86–104 nm). Particle size by continuous formation (71 nm) and the achieved encapsulation efficiency (EE) of 88% is close to the targeted parameters for Pfizer’s mRNA vaccine (66–93 nm, 88%EE). With the continuous encapsulation of nucleic acids in LNPs and the continuous production of mRNA in in vitro transcription, the basis for the holistic continuous production of mRNA is now established. We already showed that a fully autonomous process requires the incorporation of digital twins and a control strategy, with predictive process models and state-of-the-art PAT enabling real-time-release testing. This autonomous control can considerably improve productivity by about 15–20% and personnel as well as chemical reduction of about 30%. The results of this work complement this, laying the basis for fully continuous, bottleneck-free production of mRNA and other cell- and gene-therapeutic drug/vaccine candidates in a GMP- and QbD-compliant Biopharma 4.0 facilities on a flexible scale. Full article

Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic. Full article

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence. Full article

Background: COVID-19 is in its epidemic period, and China is still facing the dual risks of import and domestic rebound. To better control the COVID-19 pandemic under the existing conditions, the focus of this study is to simulate the nucleic acid testing for different population size cities in China to influence the spread of COVID-19, assess the situation under different scenarios, the demand for the laboratory testing personnel, material resources, for the implementation of the nucleic acid screening measures, emergency supplies, and the configuration of human resources to provide decision-making basis. Methods: According to the transmission characteristics of COVID-19 and the current prevention and control strategies in China, four epidemic scenarios were assumed. Based on the constructed SVEAI_iQHR model, the number of people infected with COVID-19 in cities with populations of 10 million, 5 million, and 500,000 was analyzed and predicted under the four scenarios, and the demand for laboratory testing resources was evaluated, respectively. Results: For large, medium, and small cities, whether full or regional nucleic acid screening can significantly reduce the epidemic prevention and control strategy of different scenarios laboratory testing resource demand difference is bigger, implement effective non-pharmaceutical interventions and regional nucleic acid screening measures to significantly reduce laboratory testing related resources demand, but will cause varying degrees of inspection staff shortages. Conclusion: There is still an urgent need for laboratory testing manpower in China to implement effective nucleic acid screening measures in the event of an outbreak. Cities or regions with different population sizes and levels of medical resources should flexibly implement prevention and control measures according to specific conditions after the outbreak, assess laboratory testing and human resource need as soon as possible, and prepare and allocate materials and personnel. Full article

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods—reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)—combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10–20 min, whereas for RT-LAMP, the LOD was 30–300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results. Full article