Sensitive Nucleic Acid Testing Research Articles

A one-pot loop-mediated isothermal amplification platform using fluorescent gold nanoclusters for rapid and naked-eye pathogen detection

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive nucleic acid testing method for pathogen detection, yet the absence of a straightforward readout strategy remains challenging. We've successfully designed polyethyleneimine-stabilized gold nanoclusters (PEI-AuNCs) as a cationic AuNCs indicator tailored for distinguishing LAMP results, enabling direct visual inspection under UV light. Positive LAMP reactions with PEI-AuNCs, in combination with magnesium pyrophosphate crystals, yield red-fluorescent bulk precipitates visible to the naked eye. To address contamination concerns, we introduced a one-pot reaction by incorporating AuNCs into the lid recess. This one-pot LAMP assay demonstrates exceptional detection capability, identifying Salmonella enterica at concentrations as low as 101 CFU/mL within approximately 50 min, excluding nucleic acid extraction. The platform's versatility, achieved through customizable primers, positions it as a promising molecular diagnostic tool for rapid and visual pathogen detection across scientific disciplines.

Rapid and sensitive detection of shrimp allergens within 30 min using a new food nucleic acid release agent combined with a fast real-time PCR assay

To achieve fast on-site detection of shrimp allergens in food, this study established a new direct, fast, and real-time quantitative polymerase chain reaction (PCR) technology. The technology completed nucleic acid extraction in 4 min using a new nucleic acid releaser, and the DNA samples were detected using fast qPCR within 25 min (total detection was approximately 30 min). The results indicated that the direct fast qPCR can specifically detect shrimp, with a limit of detection of 0.0001% of shrimp in artificially simulated meat samples and a detection precision variation coefficient (CV value) was less than 5%. It successfully identified shrimp in different types of commercial food samples. As a simple, fast, and sensitive nucleic acid testing technology, it has broad application prospects for the on-site detection of shrimp allergens in food.

A Portable Nucleic Acid Testing Platform with Photosensitization, a Three-Dimensionally Printed Multipiece Chip, and Digital Color Sensing.

Portable nucleic acid testing (NAT) holds great promise for point-of-care disease diagnosis and field-based applications but remains difficult to achieve. Herein, we describe a portable NAT that streamlines loop-mediated isothermal amplification with photosensitization-based color development in a fully sealed 3D-printed multipiece chip. Using a smartphone accessory and an APP, we also introduce a calibration-free quantification approach via digital color sensing and library matching. With these innovative approaches, our detection platform is highly accessible, allowing for rapid and sensitive NAT without requiring sophisticated instruments and well-trained personnel. The field applicability of our NAT platform was demonstrated by detecting tuberculosis infections in clinical sputum samples and food adulteration in commercial salmon meat products.

Fluorogenic RNA aptamer output sensors via transcription activated by recombinase polymerase amplification for nucleic acid testing

Sensitive, simple, and rapid nucleic acid testing used for pathogen detection are crucial for preventing outbreaks and transmission of infectious diseases. In this study, we present a fluorogenic RNA aptamer Output Sensors via transcription Activated by Recombinase polymerase amplification (RPA), termed ROSAR, for highly sensitive detection of DNA and RNA from pathogenic bacteria or viruses in clinical samples. The promoter primer and reporter primer facilitate the integration of the transcriptional promoter with the fluorogenic RNA aptamer transcription templates through the RPA of target DNA. Subsequently, the amplification products are transcribed by T7 RNA polymerase, producing RNA aptamer that binds to a fluorogenic dye. The assay, which can be completed within 50 min and can allow a limit of detection of level aM nucleic acid concentration, owning to the dual amplification effects of RPA and in vitro transcription. Furthermore, we develop a paper-based ROSAR by freeze-drying the reaction components, which is compatible with visual detection based on mobile phone fluorescent reader and suitable for point-of-care (POC) applications. The assay can detect various viral and bacterial nucleic acids, making it a highly promising tool for large-scale epidemic screening and rapid POC testing in home healthcare settings.

Fluorescein-switching-based lateral flow assay for the detection of microRNAs.

Lateral flow assays (LFAs) are a cost-effective and rapid colorimetric technology that can be effectively used for nucleic acid tests (NATs) in various fields such as medical diagnostics and biotechnology. Given their importance, developing more diverse LFAs that operate through novel working mechanisms is essential for designing highly selective and sensitive NATs and providing insights for designing various practical point-of-care testing (POCT) systems. Herein we report a new type of lateral flow assay (LFA) based on fluorescein-switching, enabled by nucleic acid-templated photooxidation of reduced fluorescein by riboflavin tetraacetate (RFTA). The LFA design leverages the fact that a reduced form of fluorescein, which weakly binds to gold nanoparticle (GNP)-conjugated anti-fluorescein antibodies, is oxidized in the presence of target nucleic acids to yield its native state, which then strongly binds to the antibodies. The study involved designing and optimizing probe sequences to detect miR-6090 and miR-141, which are significant markers for prostate cancer. To minimize background signals of LFAs, sodium borohydride (NaBH4) was specifically introduced as a reducing agent, and detailed procedures were established. The developed LFA system accurately identified low fmol levels of target microRNAs with minimal false positives, all detectable with the naked eye, making the system a promising tool for point-of-care diagnostics.

Single primer isothermal amplification for real-time and visual on-site detection of genetically modified crops by recognizing CP4-EPSPS gene

In response to the potential controversies on increasing circulation of genetically modified organisms (GMOs) in the global markets, the rapid and sensitive detection methods are essential for monitoring of GMOs to maintain consumers' rights. Herein, we first developed a rapid technique for the real-time and visual on-site detection of the genetically modified crops (specifically targeting CP4-EPSPS gene) by using single primer isothermal amplification (SPIA) combined with SYBR Green II. 19 GM crop events and 12 non-GM crop events were used to assess the specificity of the developed SPIA assay, which presented excellent specificity. The limit of detection of the developed SPIA method was as low as 4 copies/μL, which was better than that of the real-time PCR method (20 copies/μL). The SPIA method was further used to assess 30 of actual samples, and the experimental results revealed that 3 positive samples were found, which were in good agreement with those of the real-time PCR methods. Overall, the major advantages of the developed SPIA over real-time PCR are the lower LOD, simplicity, and inexpensiveness. The developed SPIA method provides a potential alternative for the real-time and visual on-site detection of GMOs, which also accelerates the development of highly sensitive nucleic acid testing techniques.

Relationship between SARS-CoV-2 nucleocapsid protein and N gene and its application in antigen testing kits evaluation.

More than forty antigen testing kits have been approved to response the prevalence of SARS-CoV-2 and its variant strains. However, the approved antigen testing kits are not capable of quantitative detection. Here, we successfully developed a lateral flow immunoassay based on colloidal gold nanoparticles (CGNP-based LFIA) for nucleocapsid (N) protein of SARS-CoV-2 quantitative detection. Delta strain (NMDC60042793) of SARS-CoV-2 have been cultured and analyzed by our developed digital PCR and LFIA methods to explore the relationship between N protein amount and N gene level. It indicated that the linear relationship (y=47 ×) between N protein molecule number and N gene copy number exhibited very well (R2=0.995), the virus titers and N protein amount can be roughly estimated according to nucleic acid testing. Additionally, detection limits (LODs) of nine approved antigen testing kits also have been evaluated according to the Guidelines for the registration review of 2019-nCoV antigen testing reagents. Only three antigen testing kits had LODs as stated in the instructions, the LODs of Kits have been converted into the N gene and N protein levels, according to the established relationships among virus titer vers. N gene and antigen. Results demonstrated that the sensitivity of nucleic acid testing is at least 1835 times higher than that of antigen testing. We expect that the relationship investigation and testing kits evaluation have the important directive significance to precise epidemic prevention.

Electrical Detection Assay Based on Programmable Nucleic Acid Probe for Efficient Single-Nucleotide Polymorphism Identification.

The large-scale pandemic and fast evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have triggered an urgent need for an efficient and sensitive on-site nucleic acid testing method with single-nucleotide polymorphism (SNP) identification capability. Here, we report a multiplexed electrical detection assay based on a paperclip-shaped nucleic acid probe (PNprobe) functionalized field-effect transistor (FET) biosensor for highly sensitive and specific detection and discrimination of SARS-CoV-2 variants. The three-stem structure of the PNprobe significantly amplifies the thermodynamic stability difference between variant RNAs that differ in a single-nucleotide mutation. With the assistance of combinatorial FET detection channels, the assay realizes simultaneously the detection and identification of key mutations of seven SARS-CoV-2 variants, including nucleotide substitutions and deletions at single-nucleotide resolution within 15 min. For 70 simulated throat swab samples, the multiplexed electrical detection assay shows an identification accuracy of 97.1% for the discrimination of SARS-CoV-2 variants. Our designed multiplexed electrical detection assay with SNP identification capability provides an efficient tool to achieve scalable pandemic screening.

Economics of point-of-care infant HIV tests

Economics of point-of-care infant HIV tests

CONFIRMED USE OF ANTIRETROVIRAL DRUGS FOR HIV THERAPY IN TWO BLOOD DONORS WITH POTENTIAL ELITE CONTROLLER PROFILE FROM SÃO PAULO

The use of HIV antiretroviral (ARV) as therapy -or prophylaxis by blood donors may compromises the detection ability of HIV blood screening tests and represents a risk for blood transfusion safety. Our objective was to determine the frequency in three years of undeclared ARV use by blood donors with HIV altered markers, starting in 2018 when HIV prophylaxis began in Brazil. In a retrospective cross-sectional analysis of 345.252 blood donations, 361(0.1%) blood donations were reactive for HIV antibody-antigen in a chemiluminescent immunoassay and/or RNA screening tests. Samples from 296 (81.9%) of these HIV reactive donations were available for ARV testing by high-performance liquid chromatography-tandem mass spectrometry. Confirmatory tests data recorded from donor return were assessed. Additional serological assays and/or RT-PCR were performed on donation samples of non-returned donors. The frequency of ARV use was 2/83 (2.4%) in HIV confirmed positive donors or 2/5 (40%) in potential elite controller profile donors (HIVAb/Ag-positive and HIV RNA-negative). A single ARV, lamivudine, was found in two donors at low concentrations of 11.3 and 6.7 ng/mL. The entricitabine, efavirenz, darunavir, atazanavir and tenofovir drugs were not detected in none of the 296 donations. HIV-positive individuals on unreported ARV use are donating blood and their donations jeopardize blood safety. Even though they were directly questioned about use of any medication, HIV ARVs therapy or HIV ARV prophylaxis they were not declaring accurate information in pre-donation questionnaire. We are aware that only a fraction of ARV users have been detected and we know that the greatest concern is for those on ARV prophylaxis use who may not have antibodies or RNA at detectable levels by the methods currently used. In such cases, more sensitive individual nucleic acid tests, as well as PCR that detect proviral HIV DNA in the blood, or pathogen reduction systems, may be needed. Efforts to educate blood donation candidates about the risk of unreported ARV use are needed.

Sensitivity and specificity of nucleic acid testing in close contacts of COVID-19 cases in Guangzhou

Objective: To analyze the sensitivity and specificity of SARS-CoV-2 nucleic acid testing in 20 348 close contacts of COVID-19 cases in different prevention and control stages in Guangzhou and to provide scientific evidence for optimizing epidemic response strategies. Methods: A total of 20 348 close contacts of COVID-19 cases in Guangzhou were traced between February 21 and September 22,2020. All the close contacts were tested for the nucleic acid of SARS-CoV-2. The sensitivity and specificity of nucleic acid testing and diagnosis in the different prevention and control stages were compared. Results: In 20 348 close contacts, 12 462 were males (61.24%), the median (P25,P75) of age of them was 31.0 years (23.0,43.0), the median number (P25,P75) of nucleic acid testing for them was 2.0 (1.0,3.0), and the median (P25,P75) of their quarantine days was 12.0 (8.0,13.0) days, respectively. A total of 256 COVID-19 cases were confirmed in the close contacts after seven nucleic acid tests. In the 1st, 2nd, 3rd and 7th nucleic acid testing, the sensitivity and specificity were 69.14% and 99.99% (177 cases confirmed), 89.84% and 99.99% (230 cases confirmed), 97.27% and 99.99% (249 cases confirmed), and 100.00% and 99.98%, respectively. In the three stages of COVID-19 prevention and control in China: domestic case stage, imported case stage, and imported case associated local epidemic stage, the sensitivity of the 1st nucleic acid testing was 70.68%, 68.00% and 67.35%, and the specificity was 99.98%, 100.00% and 100.00%, respectively. Conclusions: The sensitivity of nucleic acid testing in the close contacts at the different stages were consistent with slight decrease, which might be related to the increased proportion of asymptomatic infections in the late stage of epidemic prevention and control with COVID-19 in Guangzhou. It is suggested to give three nucleic acid tests to improve the sensitivity and reduce false negative risk.

A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product

BackgroundManufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. Materials and methodsThe study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. ResultsMycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. ConclusionsMycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Comparison of two digital PCR methods for EGFR DNA and SARS-CoV-2 RNA quantification

BackgroundThe COVID-19 pandemic caused by the severe acute SARS-CoV-2 virus has undeniably highlighted the importance of reliable nucleic acid quantification. Digital PCR (dPCR) is capable of the absolute quantification of nucleic acids. MethodBy using the droplet dPCR (QX200) and the digital real-time PCR (LOAA), the copy numbers were compared via multiple assays for three distinct targerts; EGFR DNA, SARS-CoV-2 and HIV-1 RNA. ResultsThe droplet dPCR and digital real-time PCR showed similar copy numbers for both DNA and RNA quantification. When the limit of detection (LOD) and limit of quantitation (LOQ) of each method were estimated for DNA and RNA targets, the digital real-time PCR showed a higher sensitivity and precision especially with low copy number targets. ConclusionThe breath of nucleic acid testing in diagnostic applications continues to expand. In this study we applied common diagnostic targets to a novel digital real-time PCR methodology. It performed comparably to the established dPCR method with distinctive advantages and disadvantages for implementing in laboratories. These rapidly developing dPCR systems can be applied to benefit the accurate and sensitive nucleic acid testing for various clinical areas.

Nucleic acid detection and quantitative analysis of influenza virus using exhaled breath condensate

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. We previously detected the nucleic acids of bacterial pathogens in EBC. Influenza viruses can be transmitted through aerosols during coughing and exhaling. Existing detection methods for influenza have various limitations. The EBC collection method is convenient, non-invasive, and reduces the risk of exposure. We investigated the detection of influenza virus in EBC using a sensitive nucleic acid testing method and performed quantitative analysis to evaluate the present and content of influenza virus in the breath. We evaluated 30 patients with respiratory tract infection during the 2019 influenza season. The clinical data and samples of nasal swabs were collected for rapid influenza diagnostic (antigen) tests. Pharyngeal swab and EBC samples were used for influenza virus nucleic acid detection. Each EBC sample was assessed twice as well as at one-month follow-up of the patients. The nucleic acid test in the EBC of 30 cases revealed 20 and two cases of influenza A and B, respectively, giving a detection rate of 73.3%. The rapid influenza diagnostic (antigen) tests revealed four and 12 cases of influenza A and B, respectively, with a detection rate of 53.3%. All pharyngeal swab samples evaluated by the nucleic acid test were influenza-positive; 12 cases were positive for both influenza A and B and 18 cases were positive for influenza B alone. The influenza viral load in the EBC was 103–107 copies ml−1. Among the 16 patients followed-up after 1 month, 4 were positive (25%) in EBC samples and 10 were positive (62.5%) in pharyngeal swab samples. It was preliminary exploration that influenza virus could be detected in EBC. EBC is one of the sample types that would be used for molecular diagnosis of influenza.

A systematic review of etiology, epidemiology, clinical manifestations, image findings, and medication of 2019 Corona Virus Disease-19 in Wuhan, China.

Background:Unknown origin pneumonia has been furiously spreading since the late of December 2019, subsequently spread to approximately all provinces and areas in China and many countries, which was announced as a Public Health Emergency of International Concern by World Health Organization (WHO). The studies on 2019 Corona Virus Disease-19 (COVID-19) conducted from various fields around the world. Herein, the objective of the present study is to summarize the etiology, epidemiology, clinical manifestations, image findings, traceability analysis, and drug development of COVID-19.Methods:The following electronic databases were searched: Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database, VIP Chinese Science and Technology Periodical Database, and Wanfang Data. Other relevant literature will be manually searched as a compliment. We have reviewed etiology, epidemiology, clinical manifestations, image findings, and medication from case reports and retrospective clinical studies relating to COVID-19 published since the outbreak.Results:The coronavirus is closely related to bat coronavirus and pangolin coronavirus. Besides, the infection pathway is confirmed to be the respiratory and digestive systems. The virus indicates person-to-person transmission and some patients present asymptomatic. The elderly have a higher mortality rate. Rapid and sensitive nucleic acid testing is usually used as a basis for diagnosis. Currently, there is no specific vaccine and antiviral drug. Intervention actions such as travel bans and quarantine adopted have effectively reduced the spread of the epidemic.Conclusion:This systemic review will provide high-quality evidence to summarize etiology, epidemiology, clinical manifestations, image findings, traceability analysis, drug development in patients with COVID-19.

HIV incidence in US first-time blood donors and transfusion risk with a 12-month deferral for men who have sex with men

AbstractIn 2015, the US Food and Drug Administration published revised guidance that recommended a change in blood donor deferral of men who have sex with men (MSM) from an indefinite to a 12-month deferral since the donor last had sex with a man. We assessed whether HIV incidence in first-time blood donors or associated transfusion risk increased. Donations in 4 major blood collection organizations were monitored for 15 months before and 2 years after implementation of the 12-month MSM deferral policy. HIV-positive donations were classified as recently acquired or long-term using a recent infection testing algorithm and incidence in both periods estimated. Residual transfusion transmission risk was estimated by multiplying incidence by the length of the infectious window period. The latter was estimated using a model based on infectious dose and the sensitivity of nucleic acid testing. Factors associated with incident infection in each period were assessed using Poisson regression. Overall HIV incidence in first-time donors before implementation of the 12-month MSM deferral was estimated at 2.62 cases per 100 000 person-years (105 PY) (95% credible interval [CI], 1.53-3.93 cases/105 PY), and after implementation at 2.85 cases/105 PY (95% CI, 1.96-3.93 cases/105 PY), with no statistically significant change. In male first-time donors, the incidence difference was 0.93 cases/105 PY (95% CI, −1.74-3.58 cases/105 PY). The residual risk of HIV transfusion transmission through components sourced from first-time donors was estimated at 0.32 transmissions per million (106) packed red blood cell transfusions (95% CI, 0.29-0.65 transmissions/106 transfusions) before and 0.35 transmissions/106 transfusions (95% CI, 0.31-0.65 transmissions/106 transfusions) after implementation. The difference was not statistically significant. Factors associated with incident infection were the same in each period. We observed no increase in HIV incidence or HIV transfusion transmission risk after implementation of a 12-month MSM deferral policy.

A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing.

The outbreak of virus-induced infectious diseases poses a global public-health challenge. Nucleic acid amplification testing (NAAT) enables early detection of pandemic viruses and plays a vital role in preventing onward transmission. However, the requirement of skilled operators, expensive instrumentation, and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients. Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive, specific, and rapid viral nucleic acid testing. The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification (RT-LAMP) were integrated into the reaction units of a microfluidic disc. The whole processing steps such as injection of reagents, fluid actuation by rotation, heating and temperature control, and detection of fluorescence signals were carried out automatically by a customized instrument. We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) armored RNA particles. The estimated limit of detection for armored RNA particles is 2 copies per reaction, the throughput is 21 reactions per disc, and the assay sample-to-answer time is approximately 70 min. This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol, and can be readily adapted for virus detection outside the diagnostic laboratory. Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s11426-020-9800-6 and is accessible for authorized users.

Paper-based microfluidics for rapid diagnostics and drug delivery

Paper is a common material that is promising for constructing microfluidic chips (lab-on-a-paper) for diagnostics and drug delivery for biomedical applications. In the past decade, extensive research on paper-based microfluidics has accumulated a large number of scientific publications in the fields of biomedical diagnosis, food safety, environmental health, drug screening and delivery. This review focuses on the recent progress on paper-based microfluidic technology with an emphasis on the design, optimization and application of the technology platform, in particular for medical diagnostics and drug delivery. Novel advances have concentrated on engineering paper devices for point-of-care (POC) diagnostics, which could be integrated with nucleic acid-based tests and isothermal amplification experiments, enabling rapid sample-to-answer assays for field testing. Among the isothermal amplification experiments, loop-mediated isothermal amplification (LAMP), an extremely sensitive nucleic acid test, specifically identifies ultralow concentrations of DNA/RNA from practical samples for diagnosing diseases. We thus mainly focus on the paper device-based LAMP assay for the rapid infectious disease diagnosis, foodborne pathogen analysis, veterinary diagnosis, plant diagnosis, and environmental public health evaluation. We also outlined progress on paper microfluidic devices for drug delivery. The paper concludes with a discussion on the challenges of this technology and our insights into how to advance science and technology towards the development of fully functional paper devices in diagnostics and drug delivery.

West Nile virus activity in United States blood donors and optimizing detection strategies: 2014-2018.

Rare transfusion-transmitted West Nile virus (WNV) cases usually occur due to gaps in testing involving converting to more sensitive nucleic acid testing (NAT) formats (referred to as triggering). Using data from 2014 to 2018, we investigated a strategy used to increase detection early in the triggering period and reviewed its yield as the individual donation (ID)-NAT geographic area was decreased. Mini-pool-NAT transitioned to ID-NAT following triggering based on one WNV NAT-reactive donation (having an elevated signal, repeat reactive, or in an area with WNV ongoing activity). ID-NAT-triggered geographic areas included an entire state (2014-2017) or collections within a 50-mile radius of the triggering donor's residential zip code (2018). During the MP- to ID-NAT transition, donation samples were retrieved and tested by ID-NAT for those with results not yet released (referred to as in-process testing). Reactive sample confirmation was performed by repeat NAT of an independent sample or antibody testing. ID-NAT included 3.2 million donations of more than 25 million tested year-round, resulting in 684 confirmed positives; all confirmed-positive donations occurred from June to December (0.64/10,000). Overall, 52% (358/684) required ID-NAT for detection, including 68 (19%) antibody negatives. Ten of 19 (53%) identified in-process were ID-NAT-only detectable, including four antibody negatives, or approximately 1 per year (2.8% of ID-NAT-only detectable). With reduced triggering geography, 12 of 19 (63%) were not identified (including 6/10 ID-NAT-only detectable, and 2/4 ID-NAT-only detectable/antibody negative). WNV NAT's utility is between June-December; however, abandoning testing outside of this time may increase risk. While in-process testing identified approximately one ID-NAT-only detectable (antibody-negative) donation per year, reducing the geographic triggered area decreased its effectiveness.

Loop-Mediated Isothermal Amplification-Coupled Glass Nanopore Counting Toward Sensitive and Specific Nucleic Acid Testing.

Solid-state nanopores have shown great promise and achieved tremendous success in label-free single-molecule analysis. However, there are three common challenges in solid-state nanopore sensors, including the nanopore size variations from batch to batch that makes the interpretation of the sensing results difficult, the incorporation of sensor specificity, and the impractical analysis time at low analyte concentration due to diffusion-limited mass transport. Here, we demonstrate a novel loop-mediated isothermal amplification (LAMP)-coupled glass nanopore counting strategy that could effectively address these challenges. By using the glass nanopore in the counting mode (versus the sizing mode), the device fabrication challenge is considerably eased since it allows a certain degree of pore size variations and no surface functionalization is needed. The specific molecule replication effectively breaks the diffusion-limited mass transport thanks to the exponential growth of the target molecules. We show the LAMP-coupled glass nanopore counting has the potential to be used in a qualitative test as well as in a quantitative nucleic acid test. This approach lends itself to most amplification strategies as long as the target template is specifically replicated in numbers. The highly sensitive and specific sensing strategy would open a new avenue for solid-state nanopore sensors toward a new form of compact, rapid, low-cost nucleic acid testing at the point of care.