A Simple, Pipette-free, and Power-free Device for Virus Nucleic Acid Detection

A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.

Value of Biofire Filmarray pneumonia panel in evaluating secondary infection in critically ill patients with Coronavirus disease 2019.

Objective To explore the application value of Biofire Filmarry pneumonia panel (PN) in detection of secondary and concomitant pathogen among critically ill patients with coronavirus disease 2019(COVID-19). Methods We consecutively included and analyzed the clinical data of critically ill patients with COVID-19 transferred to the ICU from February to April 2020 in the Sino-French Campus of Wuhan Tongji Hospital. Samples of Bronchoalveolar lavage fluid obtained by bedside bronchoscopy were sent for Biofire Filmarray PN and standard culture concomitantly. We compared the results of two methods and evaluated their concordance. Results In total, 21 critically ill patients with COVID-19 were included and 54 samples were tested, including 33 (61.1%) Biofire Filmarray PN tests (21 patients) and 21 (38.9%) standard cultures (14 patients), in which 19 pairs (38 samples) underwent both tests simultaneously. In Biofire Filmarray PN group, the turnaround time was about 1 hour. There were 74 positive results in 32 samples (97.0%) from 20 patients, including 29 cases(39.2%) of Acinetobacter baumannii complex, 21 cases (28.4%) of Pseudomonas aeruginosa, 16 cases (21.6%)of Klebsiella pneumoniae, 5 cases (6.8%) of Escherichia coli, 1 case (1.4%)each of Enterobacter cloacae, Haemophilus influenzae, and respiratory syncytial virus. In the standard culture group, the turnaround time was about 3 days. 19 positive results returned in 16 (76.2%) samples from 11 patients, including 8 cases (42.1%) of Pseudomonas aeruginosa, 6 cases (31.6%) of Acinetobacter baumannii, 4 cases (21.1%) of Stenotrophomonas malt and 1 case (5.3%) of Myxobacterium. Among the 19 pairs of "back-to-back" specimens, 15 pairs were concordant, and the agreement ratio was 78.9%. Conclusions Acinetobacter baumannii and Pseudomonas aeruginosa may be the common pathogens of secondary or concomitant infection in critically ill patients with COVID-19. Biofire Filmarray PN is a rapid diagnostic test and has application value in such patients;its sensitivity and accuracy require further investigation with larger sample sizes.Copyright © 2021, Peking Union Medical College Hospital. All rights reserved.

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection.

Loop-mediated isothermal amplification (LAMP), as the rank one alternative to a polymerase chain reaction (PCR), has been widely applied in point-of-care testing (POCT) due to its rapid, simple, and cost-effective characteristics. However, it is difficult to achieve real-time monitoring and multiplex detection with the traditional LAMP method. In addition, these approaches that use turbidimetry, sequence-independent intercalating dyes, or pH-sensitive indicators to indirectly reflect amplification can result in false-positive results if non-specific amplification occurs. To fulfill the needs of specific target detection and one-pot multiplex detection, a variety of probe-based LAMP assays have been developed. This review focuses on the principles of these assays, summarizes their applications in pathogen detection, and discusses their features and advantages over the traditional LAMP methods.

Longitudinal Monitoring of DNA Viral Loads in Transplant Patients Using Quantitative Metagenomic Next-Generation Sequencing.

INTRODUCTION: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. METHODS: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. RESULTS: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. CONCLUSIONS: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.

How to Build Live-Cell Sensor Microdevices.

There is a lot of discussion on how viruses (such as influenza and SARS-CoV-2) are transmitted in air, potentially from aerosols and respiratory droplets, and thus it is important to monitor the environment for the presence of an active pathogen. Currently, the presence of viruses is being determined using primarily nucleic acid-based detection methods, such as reverse transcription- polymerase chain reaction (RT-PCR) tests. Antigen tests have also been developed for this purpose. However, most nucleic acid and antigen methods fail to discriminate between a viable and a non-viable virus. Therefore, we present an alternative, innovative, and disruptive approach involving a live-cell sensor microdevice that captures the viruses (and bacteria) from the air, becomes infected by them, and emits signals for an early warning of the presence of pathogens. This perspective outlines the processes and components required for living sensors to monitor the presence of pathogens in built environments and highlights the opportunity to use immune sentinels in the cells of normal human skin to produce monitors for indoor air pollutants.

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection.

Seeking optimized infectious pathogen detection tools is of primary importance to lessen the spread of infections, allowing prompt medical attention for the infected. Among nucleic-acid-based sensing techniques, loop-mediated isothermal amplification is a promising method, as it provides rapid, sensitive, and specific detection of microbial and viral pathogens and has enormous potential to transform current point-of-care molecular diagnostics. In this review, the advances in LAMP-based point-of-care diagnostics assays developed during the past few years for rapid and sensitive detection of infectious pathogens are outlined. The numerous detection methods of LAMP-based biosensors are discussed in an end-point and real-time manner with ideal examples. We also summarize the trends in LAMP-on-a-chip modalities, such as classical microfluidic, paper-based, and digital LAMP, with their merits and limitations. Finally, we provide our opinion on the future improvement of on-chip LAMP methods. This review serves as an overview of recent breakthroughs in the LAMP approach and their potential for use in the diagnosis of existing and emerging diseases.

Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in northeast China.

Bovine respiratory disease complex (BRDC) involves multiple pathogens, shows diverse lung lesions, and is a major concern in calves. Pathogens from 160 lung samples of dead cattle from 81 cattle farms in northeast China from 2016 to 2021 were collected to characterize the molecular epidemiology and risk factors of BRDC and to assess the major pathogens involved in bovine suppurative or caseous necrotizing pneumonia. The BRDC was diagnosed by autopsy, pathogen isolation, PCR, or reverse transcription-PCR detection, and gene sequencing. More than 18 species of pathogens, including 491 strains of respiratory pathogens, were detected. The positivity rate of bacteria in the 160 lung samples was 31.77%, including Trueperella pyogenes (9.37%), Pasteurella multocida (8.35%), Histophilus somni (4.48%), Mannheimia haemolytica (2.44%), and other bacteria (7.13%). The positivity rate of Mycoplasma spp. was 38.9%, including M. bovis (7.74%), M. dispar (11.61%), M. bovirhinis (7.94%), M. alkalescens (6.11%), M. arginini (0.81%), and undetermined species (4.68%). Six species of viruses were detected with a positivity rate of 29.33%, including bovine herpesvirus-1 (BoHV-1; 13.25%), bovine respiratory syncytial virus (BRSV; 5.50%), bovine viral diarrhea virus (BVDV; 4.89%), bovine parainfluenza virus type-3 (BPIV-3; 4.28%), bovine parainfluenza virus type-5 (1.22%), and bovine coronavirus (2.24%). Mixed infections among bacteria (73.75%), viruses (50%), and M. bovis (23.75%) were the major features of BRDC in these cattle herds. The risk analysis for multi-pathogen co-infection indicated that BoHV-1 and H. somni; BVDV and M. bovis, P. multocida, T. pyogenes, or Mann. haemolytica; BPIV-3 and M. bovis; BRSV and M. bovis, P. multocida, or T. pyogenes; P. multocida and T. pyogenes; and M. bovis and T. pyogenes or H. somni showed co-infection trends. A survey on molecular epidemiology indicated that the occurrence rate of currently prevalent pathogens in BRDC was 46.15% (6/13) for BoHV-1.2b and 53.85% (7/13) for BoHV-1.2c, 53.3% (8/15) for BVDV-1b and 46.7% (7/15) for BVDV-1d, 29.41% (5/17) for BPIV-3a and 70.59% (12/17) for BPIV-3c, 100% (2/2) for BRSV gene subgroup IX, 91.67% (33/36) for P. multocida serotype A, and 8.33% (3/36) for P. multocida serotype D. Our research discovered new subgenotypes for BoHV-1.2c, BRSV gene subgroup IX, and P. multocida serotype D in China's cattle herds. In the BRDC cases, bovine suppurative or caseous necrotizing pneumonia was highly related to BVDV [odds ratio (OR) = 4.18; 95% confidence interval (95% CI): 1.6-10.7], M. bovis (OR = 2.35; 95% CI: 1.1-4.9), H. somni (OR = 8.2; 95% CI: 2.6-25.5) and T. pyogenes (OR = 13.92; 95% CI: 5.8-33.3). The risk factor analysis found that dairy calves <3 mo and beef calves >3 mo (OR = 5.39; 95% CI: 2.7-10.7) were more susceptible to BRDC. Beef cattle were more susceptible to bovine suppurative or caseous necrotizing pneumonia than dairy cattle (OR = 2.32; 95% CI: 1.2-4.4). These epidemiological data and the new pathogen subgenotypes will be helpful in formulating strategies of control and prevention, developing new vaccines, improving clinical differential diagnosis by necropsy, predicting the most likely pathogen, and justifying antimicrobial use.

Plasmonic nanostructure-enhanced Raman scattering for detection of SARS-CoV-2 nucleocapsid protein and spike protein variants.

Epidemiological control and public health monitoring during the outbreaks of infectious viral diseases rely on the ability to detect viral pathogens. Here we demonstrate a rapid, sensitive, and selective nanotechnology-enhanced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection based on the surface-enhanced Raman scattering (SERS) responses from the plasma-engineered, variant-specific antibody-functionalized silver microplasma-engineered nanoassemblies (AgMEN) interacting with the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. The three-dimensional (3D) porous AgMEN with plasmonic-active nanostructures provide a high sensitivity to virus detection via the remarkable SERS signal collection. Moreover, the variant-specific antibody-functionalization on the SERS-active AgMEN enabled the high selectivity of the SARS-CoV-2 S variants, including wild-type, Alpha, Delta, and Omicron, under the simulated human saliva conditions. The exceptional ultrahigh sensitivity of our SERS biosensor was demonstrated via SARS-CoV-2 S and N proteins at the detection limit of 1 fg mL-1 and 0.1 pg mL-1, respectively. Our work demonstrates a versatile SERS-based detection platform can be applied for the ultrasensitive detection of virus variants, infectious diseases, and cancer biomarkers.

Application of the CRISPR/Cas System in Pathogen Detection: A Review.

Early and rapid diagnosis of pathogens is important for the prevention and control of epidemic disease. The polymerase chain reaction (PCR) technique requires expensive instrument control, a special test site, complex solution treatment steps and professional operation, which can limit its application in practice. The pathogen detection method based on the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated protein (CRISPR/Cas) system is characterized by strong specificity, high sensitivity and convenience for detection, which is more suitable for practical applications. This article first reviews the CRISPR/Cas system, and then introduces the application of the two types of systems represented by Type II (cas9), Type V (cas12a, cas12b, cas14a) and Type VI (cas13a) in pathogen detection. Finally, challenges and prospects are proposed.

A NOVEL STRATEGY FOR POWER-FREE READOUT OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION USING POLYDOPAMINE INTEGRATED INTO A PAPER DEVICE FOR PATHOGEN DETECTION

The result readouts of loop-mediated isothermal amplification (LAMP) still remain challenging because current techniques require bulky equipment and could not give clear visualization. In this study, we developed a paper device to integrate LAMP and a novel strategy for power-free and naked-eye readout of result relied on polydopamine aggregation. The introduced paper device was used to detect DNA extracted from Escherichia coli O157:H7 (E. coli O157:H7), Enterococcus faecium (E. faecium), and SARS-CoV-2 plasmid. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Pathogen detection and characterization from throat swabs using unbiased metatranscriptomic analyses.

OBJECTIVES: Infectious diseases are common but are not easily or readily diagnosed with current methodologies. This problem is further exacerbated by the constant presence of mutated, emerging, and novel pathogens. One of the most common sites of infection by many pathogens is the human throat. However, there is no universal diagnostic test that can distinguish these pathogens. Metatranscriptomic (MT) analysis of the throat represents an important and novel development in infectious disease detection and characterization, because it is able to identify all pathogens using a fully unbiased approach. METHODS: To test the utility of an MT approach to pathogen detection, throat samples were collected from participants before, during, and after an acute sickness. RESULTS: Clear sickness-associated shifts in pathogenic microorganisms were detected in the patients. Important insights into microbial functions and antimicrobial resistance genes were obtained. CONCLUSION: MT analysis of the throat represents an effective method for the unbiased identification and characterization of pathogens. Because MT data include all microorganisms in the sample, this approach should not only allow the identification of pathogens, but provide an understanding of the effects of the resident throat microbiome in the context of human health and disease.

Applications of Nanozymology in the Detection and Identification of Viral, Bacterial and Fungal Pathogens.

Nanozymes are synthetic nanoparticulate materials that mimic the biological activities of enzymes by virtue of their surface chemistry. Enzymes catalyze biological reactions with a very high degree of specificity. Examples include the horseradish peroxidase, lactate, glucose, and cholesterol oxidases. For this reason, many industrial uses of enzymes outside their natural environments have been developed. Similar to enzymes, many industrial applications of nanozymes have been developed and used. Unlike the enzymes, however, nanozymes are cost-effectively prepared, purified, stored, and reproducibly and repeatedly used for long periods of time. The detection and identification of pathogens is among some of the reported applications of nanozymes. Three of the methodologic milestones in the evolution of pathogen detection and identification include the incubation and growth, immunoassays and the polymerase chain reaction (PCR) strategies. Although advances in the history of pathogen detection and identification have given rise to novel methods and devices, these are still short of the response speed, accuracy and cost required for point-of-care use. Debuting recently, nanozymology offers significant improvements in the six methodological indicators that are proposed as being key in this review, including simplicity, sensitivity, speed of response, cost, reliability, and durability of the immunoassays and PCR strategies. This review will focus on the applications of nanozymes in the detection and identification of pathogens in samples obtained from foods, natural, and clinical sources. It will highlight the impact of nanozymes in the enzyme-linked immunosorbent and PCR strategies by discussing the mechanistic improvements and the role of the design and architecture of the nanozyme nanoconjugates. Because of their contribution to world health burden, the three most important pathogens that will be considered include viruses, bacteria and fungi. Although not quite seen as pathogens, the review will also consider the detection of cancer cells and helminth parasites. The review leaves very little doubt that nanozymology has introduced remarkable advances in enzyme-linked immunosorbent assays and PCR strategies for detecting these five classes of pathogens. However, a gap still exists in the application of nanozymes to detect and identify fungal pathogens directly, although indirect strategies in which nanozymes are used have been reported. From a mechanistic point of view, the nanozyme technology transfer to laboratory research methods in PCR and enzyme-linked immunosorbent assay studies, and the point-of-care devices such as electronic biosensors and lateral flow detection strips, that is currently taking place, is most likely to give rise to no small revolution in each of the six methodological indicators for pathogen detection and identification. While the evidence of widespread research reports, clinical trials and point-of-care device patents support this view, the gaps that still exist point to a need for more basic research studies to be conducted on the applications of nanozymology in pathogen detection and identification. The multidisciplinary nature of the research on the application of nanozymes in the detection and identification of pathogens requires chemists and physicists for the design, fabrication, and characterization of nanozymes; microbiologists for the design, testing and analysis of the methodologies, and clinicians or clinical researchers for the evaluation of the methodologies and devices in the clinic. Many reports have also implicated required skills in mathematical modelling, and electronic engineering. While the review will conclude with a synopsis of the impact of nanozymology on the detection and identification of viruses, bacteria, fungi, cancer cells, and helminths, it will also point out opportunities that exist in basic research as well as opportunities for innovation aimed at novel laboratory methodologies and devices. In this regard there is no doubt that there are numerous unexplored research areas in the application of nanozymes for the detection of pathogens. For example, most research on the applications of nanozymes for the detection and identification of fungi is so far limited only to the detection of mycotoxins and other chemical compounds associated with fungal infection. Therefore, there is scope for exploration of the application of nanozymes in the direct detection of fungi in foods, especially in the agricultural production thereof. Many fungal species found in seeds severely compromise their use by inactivating the germination thereof. Fungi also produce mycotoxins that can severely compromise the health of humans if consumed.

Wastewater treatment plant operators report high capacity to support wastewater surveillance for COVID-19 across New York State, USA.

Wastewater surveillance for infectious disease expanded greatly during the COVID-19 pandemic. As a collaboration between sanitation engineers and scientists, the most cost-effective deployment of wastewater surveillance routinely tests wastewater samples from wastewater treatment plants. To evaluate the capacity of treatment plants of different sizes and characteristics to participate in surveillance efforts, we developed and distributed a survey to New York State municipal treatment plant supervisors in the summer and fall of 2021. The goal of the survey was to assess the knowledge, capacity, and attitudes toward wastewater surveillance as a public health tool. Our objectives were to: (1) determine what treatment plant operators know about wastewater surveillance for public health; (2) assess how plant operators feel about the affordability and benefits of wastewater surveillance; and (3) determine how frequently plant personnel can take and ship samples using existing resources. Results show that 62% of respondents report capacity to take grab samples twice weekly. Knowledge about wastewater surveillance was mixed with most supervisors knowing that COVID-19 can be tracked via wastewater but having less knowledge about surveillance for other public health issues such as opioids. We found that attitudes toward wastewater testing for public health were directly associated with differences in self-reported capacity of the plant to take samples. Further, findings suggest a diverse capacity for sampling across sewer systems with larger treatment plants reporting greater capacity for more frequent sampling. Findings provide guidance for outreach activities as well as important insight into treatment plant sampling capacity as it is connected to internal factors such as size and resource availability. These may help public health departments understand the limitations and ability of wastewater surveillance for public health benefit.

Dogs Detecting COVID-19 From Sweat and Saliva of Positive People: A Field Experience in Mexico.

Context: Molecular tests are useful in detecting COVID-19, but they are expensive in developing countries. COVID-19-sniffing dogs are an alternative due to their reported sensitivity (>80%) and specificity (>90%). However, most of the published evidence is experimental, and there is a need to determine the performance of the dogs in field conditions. Hence, we aimed to test the sensitivity and specificity of COVID-19-sniffing dogs in the field. Methods: We trained four dogs with sweat and three dogs with saliva of COVID-19-positive patients, respectively, for 4.5 months. The samples were obtained from a health center in Hermosillo, Sonora, with the restriction to spend 5 min per patient. We calculated sensitivity, specificity, and their 95% confidence intervals (CI). Results: Two sweat-sniffing dogs reached 76 and 80% sensitivity, with the 95% CI not overlapping the random value of 50%, and 75 and 88% specificity, with the 95% CI not overlapping the 50% value. The 95% CI of the sensitivity and specificity of the other two sweat dogs overlapped the 50% value. Two saliva-sniffing dogs had 70 and 78% sensitivity, and the 95% CI of their sensitivity and specificity did not overlap the 50% value. The 95% CI of the third dog's sensitivity and specificity overlapped the 50% value. Conclusion: Four of the six dogs were able to detect positive samples of patients with COVID-19, with sensitivity and specificity values significantly different from random in the field. We considered the performance of the dogs promising because it is reasonable to expect that with gauze exposed for a longer time to sweat and saliva of people with COVID-19, their detection capacity would improve. The target is to reach the sensitivity range requested by the World Health Organization for the performance of an antigen test (≥80% sensitivity, ≥97% specificity). If so, dogs could become important allies for the control of the COVID-19 pandemic, especially in developing countries.

Detection and isolation of airborne SARS-CoV-2 in a hospital setting.

Transmission mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are incompletely understood. In particular, aerosol transmission remains unclear, with viral detection in air and demonstration of its infection potential being actively investigated. To this end, we employed a novel electrostatic collector to sample air from rooms occupied by COVID-19 patients in a major Swedish hospital. Electrostatic air sampling in conjunction with extraction-free, reverse-transcriptase polymerase chain reaction (hid-RT-PCR) enabled detection of SARS-CoV-2 in air from patient rooms (9/22; 41%) and adjoining anterooms (10/22; 45%). Detection with hid-RT-PCR was concomitant with viral RNA presence on the surface of exhaust ventilation channels in patients and anterooms more than 2 m from the COVID-19 patient. Importantly, it was possible to detect active SARS-CoV-2 particles from room air, with a total of 496 plaque-forming units (PFUs) being isolated, establishing the presence of infectious, airborne SARS-CoV-2 in rooms occupied by COVID-19 patients. Our results support circulation of SARS-CoV-2 via aerosols and urge the revision of existing infection control frameworks to include airborne transmission.

Performance of Five Metagenomic Classifiers for Virus Pathogen Detection Using Respiratory Samples from a Clinical Cohort.

Viral metagenomics is increasingly applied in clinical diagnostic settings for detection of pathogenic viruses. While several benchmarking studies have been published on the use of metagenomic classifiers for abundance and diversity profiling of bacterial populations, studies on the comparative performance of the classifiers for virus pathogen detection are scarce. In this study, metagenomic data sets (n = 88) from a clinical cohort of patients with respiratory complaints were used for comparison of the performance of five taxonomic classifiers: Centrifuge, Clark, Kaiju, Kraken2, and Genome Detective. A total of 1144 positive and negative PCR results for a total of 13 respiratory viruses were used as gold standard. Sensitivity and specificity of these classifiers ranged from 83 to 100% and 90 to 99%, respectively, and was dependent on the classification level and data pre-processing. Exclusion of human reads generally resulted in increased specificity. Normalization of read counts for genome length resulted in a minor effect on overall performance, however it negatively affected the detection of targets with read counts around detection level. Correlation of sequence read counts with PCR Ct-values varied per classifier, data pre-processing (R2 range 15.1-63.4%), and per virus, with outliers up to 3 log10 reads magnitude beyond the predicted read count for viruses with high sequence diversity. In this benchmarking study, sensitivity and specificity were within the ranges of use for diagnostic practice when the cut-off for defining a positive result was considered per classifier.

Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19.

To explore the application value of nanopore sequencing technique in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID-19). A total of 77 clinical specimens from 3 patients with severe COVID-19 were collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included , , and , et al. , , were also detected in clinical microbiological culture-based detection; was detected in respiratory pathogen screening qPCR panel; was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against , and the infection was controlled. Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

A review on the recent achievements on coronaviruses recognition using electrochemical detection methods.

Various coronaviruses, which cause a wide range of human and animal diseases, have emerged in the past 50 years. This may be due to their abilities to recombine, mutate, and infect multiple species and cell types. A novel coronavirus, which is a family of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), has been termed COVID-19 by the World Health Organization (WHO). COVID-19 is the strain that has not been previously identified in humans. The early identification and diagnosis of the virus is crucial for effective pandemic prevention. In this study, we review shortly various diagnostic methods for virus assay and focus on recent advances in electrochemical biosensors for COVID-19 detection.

Fabrication of a fully integrated paper microdevice for point-of-care testing of infectious disease using Safranin O dye coupled with loop-mediated isothermal amplification.

In this study, we introduce a paper microdevice fully integrating DNA extraction, loop-mediated isothermal amplification (LAMP), and Safranin O-based colorimetric detection of two major infectious pathogens, namely SARS-CoV-2 and Enterococcus faecium. The paper microdevice is composed of two parts: sample and reaction chambers. A sealing film acted as a bottom layer to allow foldable motion for transferring DNA from sample chamber to reaction chamber in a seamless manner. An FTA card was employed in the sample chamber for DNA extraction and purification from bacteria-spiked milk. After LAMP reaction at 65 °C for 30 min, a novel aggregation-based DNA detection was obtained by Safranin O polymerization in the reaction chamber. Specifically, Safranin O underwent polymerization by addition of oxidant to form Safranin O oligomers. The electrostatic interaction between the positively charged Safranin O oligomers and the negatively charged DNA comprising LAMP amplicons resulted in the aggregation with a dark red color. Meanwhile, in the absence of LAMP amplicons, Safranin O oligomers were well dispersed and displayed their original red color. By using Safranin O-based detection, SARS-CoV-2 and E. faecium were successfully identified by naked eye within 60 min, and the limits of detection were 10-4 ng/µL and 102 CFU/mL, respectively. These results indicate that a fully integrated paper microdevice plays an important role in sample-in-answer-out format in the genetic analyses of infectious disease and serves as a rapid tool for controlling the spread of diseases.

Recombinase polymerase amplification integrated with microfluidics for nucleic acid testing at point of care.

Nucleic acid testing (NAT) implemented on a portable, miniaturized, and integrated device with rapid and sensitive results readout is highly demanded for pathogen detection or genetic screening at resource-limited settings, especially after the outbreak of coronavirus disease 2019 (COVID-19). The integration of recombinase polymerase amplification (RPA) with emerging microfluidics, classified by paper-based microfluidics and chip-based microfluidics, shows great potential to perform laboratory independent NAT assays at point of care with minimal labor, time and energy consumption. This review summarizes the state-of-the-art of RPA integrated with paper-based microfluidics and chip-based microfluidics, and discusses their pros and cons. Finally, existing challenges and possible ways for optimization of microfluidics-based RPA are proposed.