scholarly journals Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Padmapriya P. Banada ◽  
Srinidhi Deshpande ◽  
Sukalyani Banik ◽  
Darshini Shah ◽  
Ranie Koshy ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Severe Disease ◽  
High Sensitivity ◽  
Negative Control ◽  
Blood Samples ◽  
Content Type ◽  
Clinical Patient

ABSTRACT Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis. The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

scholarly journals Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

2017 ◽  
Vol 55 (10) ◽  
pp. 2964-2971 ◽  
Author(s):  
Padmapriya P. Banada ◽  
Srinidhi Deshpande ◽  
Riccardo Russo ◽  
Eric Singleton ◽  
Darshini Shah ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Severe Disease ◽  
Automated System ◽  
Bacterial Isolates ◽  
Sample Processing ◽  
Blood Samples ◽  
Content Type

ABSTRACT Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non- B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log 10 dynamic range. Assay specificity was 100% for tests of non- B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

scholarly journals Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System

2016 ◽  
Vol 55 (1) ◽  
pp. 291-301 ◽  
Author(s):  
Padmapriya P. Banada ◽  
Srinidhi Deshpande ◽  
Soumitesh Chakravorty ◽  
Riccardo Russo ◽  
James Occi ◽  
...  
Keyword(s):  
Blood Culture ◽  
Francisella Tularensis ◽  
Whole Blood ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Bloodstream Infections ◽  
Content Type ◽  
Testing Procedures ◽  
Point Of Care Detection

ABSTRACTFrancisella tularensisis a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitiveF. tularensisassay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system.F. tularensisDNA in buffer or CFU ofF. tularensiswas spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected withF. tularensisSchu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/mlF. tularensisin both human and macaque blood. In infected macaques, the assay detectedF. tularensison days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detectF. tularensisin bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.

scholarly journals Ag nanoparticles outperform Au nanoparticles for the use as label in electrochemical point-of-care sensors

2021 ◽  
Author(s):  
Franziska Beck ◽  
Carina Horn ◽  
Antje J. Baeumner
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Automatic Measurement ◽  
High Sensitivity ◽  
Differential Pulse ◽  
Small Sample ◽  
Blood Protein ◽  
User Friendliness ◽  
Response Curves ◽  
Assay Protocol

AbstractElectrochemical immunosensors enable rapid analyte quantification in small sample volumes, and have been demonstrated to provide high sensitivity and selectivity, simple miniaturization, and easy sensor production strategies. As a point-of-care (POC) format, user-friendliness is equally important and most often not combinable with high sensitivity. As such, we demonstrate here that a sequence of metal oxidation and reduction, followed by stripping via differential pulse voltammetry (DPV), provides lowest limits of detection within a 2-min automatic measurement. In exchanging gold nanoparticles (AuNPs), which dominate in the development of POC sensors, with silver nanoparticles (AgNPs), not only better sensitivity was obtained, but more importantly, the assay protocol could be simplified to match POC requirements. Specifically, we studied both nanoparticles as reporter labels in a sandwich immunoassay with the blood protein biomarker NT-proBNP. For both kinds of nanoparticles, the dose-response curves easily covered the ng∙mL−1 range. The mean standard deviation of all measurements of 17% (n ≥ 4) and a limit of detection of 26 ng∙mL−1 were achieved using AuNPs, but their detection requires addition of HCl, which is impossible in a POC format. In contrast, since AgNPs are electrochemically less stable, they enabled a simplified assay protocol and provided even lower LODs of 4.0 ng∙mL−1 in buffer and 4.7 ng∙mL−1 in human serum while maintaining the same or even better assay reliability, storage stability, and easy antibody immobilization protocols. Thus, in direct comparison, AgNPs clearly outperform AuNPs in desirable POC electrochemical assays and should gain much more attention in the future development of such biosensors.

scholarly journals Direct-RT-qPCR Detection of SARS-CoV-2 without RNA Extraction as Part of a COVID-19 Testing Strategy: From Sample to Result in One Hour

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 605 ◽  
Author(s):  
Eva Kriegova ◽  
Regina Fillerova ◽  
Petr Kvapil
Keyword(s):  
High Throughput Screening ◽  
High Speed ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
Ease Of Use ◽  
Diagnostic Tools ◽  
Heat Inactivation

Due to the lack of protective immunity in the general population and the absence of effective antivirals and vaccines, the Coronavirus disease 2019 (COVID-19) pandemic continues in some countries, with local epicentres emerging in others. Due to the great demand for effective COVID-19 testing programmes to control the spread of the disease, we have suggested such a testing programme that includes a rapid RT-qPCR approach without RNA extraction. The Direct-One-Step-RT-qPCR (DIOS-RT-qPCR) assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one hour while maintaining the high sensitivity and specificity required of diagnostic tools. This optimised protocol allows for the direct use of swab transfer media (14 μL) without the need for RNA extraction, achieving comparable sensitivity to the standard method that requires the time-consuming and costly step of RNA isolation. The limit of detection for DIOS-RT-qPCR was lower than seven copies/reaction, which translates to 550 virus copies/mL of swab. The speed, ease of use and low price of this assay make it suitable for high-throughput screening programmes. The use of fast enzymes allows RT-qPCR to be performed under standard laboratory conditions within one hour, making it a potential point-of-care solution on high-speed cycling instruments. This protocol also implements the heat inactivation of SARS-CoV-2 (75 °C for 10 min), which renders samples non-infectious, enabling testing in BSL-2 facilities. Moreover, we discuss the critical steps involved in developing tests for the rapid detection of COVID-19. Implementing rapid, easy, cost-effective methods can help control the worldwide spread of the COVID-19 infection.

scholarly journals Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

2015 ◽  
Vol 81 (9) ◽  
pp. 3039-3048 ◽  
Author(s):  
Stefanie Rettcher ◽  
Felicitas Jungk ◽  
Christoph Kühn ◽  
Hans-Joachim Krause ◽  
Greta Nölke ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Plant Pathogens ◽  
High Sensitivity ◽  
Potato Virus X ◽  
Plant Viruses ◽  
Economic Losses ◽  
Virus Concentration ◽  
Content Type ◽  
Agricultural Industry ◽  
Sum Frequency

ABSTRACTPlant pathogens cause major economic losses in the agricultural industry because late detection delays the implementation of measures that can prevent their dissemination. Sensitive and robust procedures for the rapid detection of plant pathogens are therefore required to reduce yield losses and the use of expensive, environmentally damaging chemicals. Here we describe a simple and portable system for the rapid detection of viral pathogens in infected plants based on immunofiltration, subsequent magnetic detection, and the quantification of magnetically labeled virus particles.Grapevine fanleaf virus(GFLV) was chosen as a model pathogen. Monoclonal antibodies recognizing the GFLV capsid protein were immobilized onto immunofiltration columns, and the same antibodies were linked to magnetic nanoparticles. GFLV was quantified by immunofiltration with magnetic labeling in a double-antibody sandwich configuration. A magnetic frequency mixing technique, in which a two-frequency magnetic excitation field was used to induce a sum frequency signal in the resonant detection coil, corresponding to the virus concentration within the immunofiltration column, was used for high-sensitivity quantification. We were able to measure GFLV concentrations in the range of 6 ng/ml to 20 μg/ml in less than 30 min. The magnetic immunoassay could also be adapted to detect other plant viruses, includingPotato virus XandTobacco mosaic virus, with detection limits of 2 to 60 ng/ml.

scholarly journals Whole Blood Immunoassay Based on Centrifugal Bead Sedimentation

2011 ◽  
Vol 57 (5) ◽  
pp. 753-761 ◽  
Author(s):  
Ulrich Y Schaff ◽  
Greg J Sommer
Keyword(s):  
Whole Blood ◽  
Single Channel ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Microfluidic System ◽  
Blood Samples ◽  
Reactive Protein ◽  
Immunoassay Technique ◽  
Microfluidic Immunoassay

BACKGROUND Centrifugal “lab on a disk” microfluidics is a promising avenue for developing portable, low-cost, automated immunoassays. However, the necessity of incorporating multiple wash steps results in complicated designs that increase the time and sample/reagent volumes needed to run assays and raises the probability of errors. We present proof of principle for a disk-based microfluidic immunoassay technique that processes blood samples without conventional wash steps. METHODS Microfluidic disks were fabricated from layers of patterned, double-sided tape and polymer sheets. Sample was mixed on-disk with assay capture beads and labeling antibodies. Following incubation, the assay beads were physically separated from the blood cells, plasma, and unbound label by centrifugation through a density medium. A signal-laden pellet formed at the periphery of the disk was analyzed to quantify concentration of the target analyte. RESULTS To demonstrate this technique, the inflammation biomarkers C-reactive protein and interleukin-6 were measured from spiked mouse plasma and human whole blood samples. On-disk processing (mixing, labeling, and separation) facilitated direct assays on 1-μL samples with a 15-min sample-to-answer time, <100 pmol/L limit of detection, and 10% CV. We also used a unique single-channel multiplexing technique based on the sedimentation rate of different size or density bead populations. CONCLUSIONS This portable microfluidic system is a promising method for rapid, inexpensive, and automated detection of multiple analytes directly from a drop of blood in a point-of-care setting.

scholarly journals Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Lars D. Renner ◽  
Jindong Zan ◽  
Linda I. Hu ◽  
Manuel Martinez ◽  
Pedro J. Resto ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Bacterial Pathogens ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
Technology Gap ◽  
Content Type ◽  
Antimicrobial Drug Resistance ◽  
Industrial Processing

ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

scholarly journals Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Alison Burdett ◽  
Christofer Toumazou ◽  
Rashmita Sahoo ◽  
Adam Mujan ◽  
Tsz-Kin Hon ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
High Sensitivity ◽  
Pool Size ◽  
Pooled Testing ◽  
Paired Samples ◽  
Absolute Strength ◽  
Potential Benefits ◽  
First Time ◽  
Multiple Patients

Abstract Background As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients’ samples. Method We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals’ sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. Results Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0–60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6–56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13–60 vs. pooled sputum MAR 25.3, SD 14.6, range 1–54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12–3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. Conclusion We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population “bubble”.

scholarly journals Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia

2020 ◽  
Vol 66 (4) ◽  
pp. 549-555 ◽  
Author(s):  
Daniel K W Chu ◽  
Yang Pan ◽  
Samuel M S Cheng ◽  
Kenrie P Y Hui ◽  
Pavithra Krishnan ◽  
...  
Keyword(s):  
Early Identification ◽  
Rapid Detection ◽  
Dynamic Range ◽  
Positive Control ◽  
Negative Control ◽  
Sars Coronavirus ◽  
Reverse Transcription Pcr ◽  
Molecular Tests ◽  
Novel Coronavirus ◽  
Negative Controls

Abstract Background A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. The outbreak was initially detected in Wuhan, a major city of China, but has subsequently been detected in other provinces of China. Travel-associated cases have also been reported in a few other countries. Outbreaks in health care workers indicate human-to-human transmission. Molecular tests for rapid detection of this virus are urgently needed for early identification of infected patients. Methods We developed two 1-step quantitative real-time reverse-transcription PCR assays to detect two different regions (ORF1b and N) of the viral genome. The primer and probe sets were designed to react with this novel coronavirus and its closely related viruses, such as SARS coronavirus. These assays were evaluated using a panel of positive and negative controls. In addition, respiratory specimens from two 2019-nCoV-infected patients were tested. Results Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10−4-2000 TCID50/reaction). Using DNA plasmids as positive standards, the detection limits of these assays were found to be below 10 copies per reaction. All negative control samples were negative in the assays. Samples from two 2019-nCoV-infected patients were positive in the tests. Conclusions The established assays can achieve a rapid detection of 2019n-CoV in human samples, thereby allowing early identification of patients.

scholarly journals Recent Advances of Field-Effect Transistor Technology for Infectious Diseases

Biosensors ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 103
Author(s):  
Abbas Panahi ◽  
Deniz Sadighbayan ◽  
Saghi Forouhi ◽  
Ebrahim Ghafar-Zadeh
Keyword(s):  
Infectious Diseases ◽  
Field Effect ◽  
Field Effect Transistor ◽  
Limit Of Detection ◽  
Point Of Care ◽  
High Sensitivity ◽  
Cmos Technology ◽  
Oxide Semiconductor ◽  
Label Free ◽  
Effect Transistor

Field-effect transistor (FET) biosensors have been intensively researched toward label-free biomolecule sensing for different disease screening applications. High sensitivity, incredible miniaturization capability, promising extremely low minimum limit of detection (LoD) at the molecular level, integration with complementary metal oxide semiconductor (CMOS) technology and last but not least label-free operation were amongst the predominant motives for highlighting these sensors in the biosensor community. Although there are various diseases targeted by FET sensors for detection, infectious diseases are still the most demanding sector that needs higher precision in detection and integration for the realization of the diagnosis at the point of care (PoC). The COVID-19 pandemic, nevertheless, was an example of the escalated situation in terms of worldwide desperate need for fast, specific and reliable home test PoC devices for the timely screening of huge numbers of people to restrict the disease from further spread. This need spawned a wave of innovative approaches for early detection of COVID-19 antibodies in human swab or blood amongst which the FET biosensing gained much more attention due to their extraordinary LoD down to femtomolar (fM) with the comparatively faster response time. As the FET sensors are promising novel PoC devices with application in early diagnosis of various diseases and especially infectious diseases, in this research, we have reviewed the recent progress on developing FET sensors for infectious diseases diagnosis accompanied with a thorough discussion on the structure of Chem/BioFET sensors and the readout circuitry for output signal processing. This approach would help engineers and biologists to gain enough knowledge to initiate their design for accelerated innovations in response to the need for more efficient management of infectious diseases like COVID-19.

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