scholarly journals Rapid Detection of Hepatitis B Virus in Blood Samples Using a Combination of Polymerase Spiral Reaction With Nanoparticles Lateral-Flow Biosensor

2021 ◽  
Vol 7 ◽  
Author(s):  
Lin Lin ◽  
Jinshuai Guo ◽  
Haiyang Liu ◽  
Xiaofeng Jiang
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Technique ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Blood Samples ◽  
Nucleic Acid Amplification Technology

A rapid, highly sensitive, and robust diagnostic technique for point-of-care (PoC) testing can be developed using the combination of the nanoparticle-based lateral flow biosensors (LFB) and isothermal nucleic acid amplification technology. Here, we developed a polymerase spiral reaction (PSR) containing FITC-labeled DNA probes coupled with the nanoparticle-based LFB assay (PSR-LFB) to detect the amplified products to detect HBV visually. Under the optimized conditions, the PSR assay involved incubation of the reaction mixture for 20 min at 63°C, followed by visual detection of positive amplicons using LFB, which would generate a red test line based on the biotin/streptavidin interaction and immunoreactions, within 5 min. A cross-reactivity test revealed that the developed PSR-LFB assay showed good specificity for HBV and could distinguish HBV from other pathogenic microorganisms. For the analytical sensitivity, the limit of detection (LoD) of PSR-LFB assay was recorded as 5.4 copies/mL of HBV genomic DNA, which was ten-times more sensitive than qPCR and loop-mediated isothermal amplification (LAMP). Additionally, all the HBV-positive (29/82) samples, identified using ELISA, were also successfully detected by the PSR-LFB assay. We found that the true positive rate of the PSR-LFB assay was higher than that of qPCR (100 vs. 89.66%, respectively), as well as the LAMP assay (100 vs. 96.55%, respectively). Furthermore, the integrated procedure could be completed in 60 min, including the processing of the blood samples (30 min), an isothermal reaction (20 min), and result visualization (5 min). Thus, this PSR-LFB assay could be a potentially useful technique for PoC diagnosis of HBV in resource-limited countries.

scholarly journals Multiple Bacteria Identification in the Point-of-Care: an Old Method Serving a New Approach

Sensors ◽  
2020 ◽  
Vol 20 (12) ◽  
pp. 3351
Author(s):  
Sara Viveiros ◽  
Mónica Rodrigues ◽  
Débora Albuquerque ◽  
Sofia A. M. Martins ◽  
Susana Cardoso ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Bovine Mastitis ◽  
Cross Reactivity ◽  
Superparamagnetic Nanoparticles ◽  
Nucleic Acid Amplification ◽  
Streptococcus Uberis ◽  
Detection Assay

The accurate diagnosis of bacterial infections is of critical importance for effective treatment decisions. Due to the multietiologic nature of most infectious diseases, multiplex assays are essential for diagnostics. However, multiplexability in nucleic acid amplification-based methods commonly resorts to multiple primers and/or multiple reaction chambers, which increases analysis cost and complexity. Herein, a polymerase chain reaction (PCR) offer method based on a universal pair of primers and an array of specific oligonucleotide probes was developed through the analysis of the bacterial 16S ribosomal RNA gene. The detection system consisted of DNA hybridization over an array of magnetoresistive sensors in a microfabricated biochip coupled to an electronic reader. Immobilized probes interrogated single-stranded biotinylated amplicons and were obtained using asymmetric PCR. Moreover, they were magnetically labelled with streptavidin-coated superparamagnetic nanoparticles. The benchmarking of the system was demonstrated to detect five major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. All selected probes proved to specifically detect their respective amplicon without significant cross reactivity. A calibration curve was performed for S. agalactiae, which demonstrates demonstrating a limit of detection below 30 fg/µL. Thus, a sensitive and specific multiplex detection assay was established, demonstrating its potential as a bioanalytical device for point-of-care applications.

scholarly journals Development and Evaluation of Europium-Based Quantitative Lateral Flow Immunoassay for the Chronic Kidney Disease Marker Cystatin-C

2021 ◽  
Author(s):  
SATHEESH NATARAJAN ◽  
Ebru saatci
Keyword(s):  
Chronic Kidney Disease ◽  
Kidney Disease ◽  
Cystatin C ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Lateral Flow ◽  
Fluorescence Measurement ◽  
Analytical Sensitivity ◽  
Linear Calibration ◽  
Time Resolved

Abstract This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. Some Cystatin-c immunoassays are sensitive, accurate, and available for clinical application, but they are expensive and time-consuming procedures. Also, conventional organic dye-based fluorescence lateral flow assay showed more background fluorescence interference and low analytical sensitivity. So this Europium-based sandwich immunoassay was developed to detect the concentration of cystatin-c in a urine sample with captured anti-cystatin-c antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-cystatin-c labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 minutes. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015-32 µg/ml, and the limit of detection (LOD) quantified was 0.0001µg/ml. This current work has improved the LOD of our previous work from 0.013µg/ml to 0.001µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine

scholarly journals Development of a highly sensitive magneto-enzyme lateral flow immunoassay for dengue NS1 detection

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7779 ◽  
Author(s):  
Tien V. Tran ◽  
Ba V. Nguyen ◽  
Thao T.P. Nguyen ◽  
Tung T. Tran ◽  
Khanh G. Pham ◽  
...  
Keyword(s):  
Test Performance ◽  
Limit Of Detection ◽  
Dengue Infection ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
The Novel ◽  
Structural Protein ◽  
Highly Sensitive

Background Dengue infection represents a global health issue of growing importance. Dengue non-structural protein 1 (NS1) plays a central role in the early detection of the disease. The most common method for NS1 detection is testing by lateral flow immunoassays (LFIAs) with varying sensitivity. In this study, we present a highly sensitive magneto-enzyme LFIA for prompt diagnosis of dengue. Methods We have demonstrated the development of a magneto-enzyme LFIA combining super-paramagnetic nanoparticles as labels and Biotin–Streptavidin signal amplification strategy to detect dengue NS1. Factors affecting the test performance including antibody pair, super-paramagnetic nanoparticle size, nitrocellulose membrane type, amounts of detection and capture antibodies, and amounts of Streptavidin-polyHRP were optimized. Analytical sensitivity and cross-reactivity were determined. Clinical performance of the novel assay was evaluated using a panel of 120 clinical sera. Results This newly developed assay could detect NS1 of all four serotypes of dengue virus (DENV). The limit of detection (LOD) was found to be as low as 0.25 ng ml−1 for DENV-1 and DENV-3, 0.1 ng ml−1 for DENV-2, and 1.0 ng ml−1 for DENV-4. The LOD for DENV-2 was a 50-fold improvement over the best values previously reported. There was an absence of cross-reactivity with Zika NS1, Hepatitis B virus, Hepatitis C virus, and Japanese encephalitis virus. The sensitivity and specificity of the novel assay were 100% when tested on clinical samples. Conclusions We have successfully developed a magneto-enzyme LFIA, allowing rapid and highly sensitive detection of dengue NS1, which is essential for proper management of patients infected with DENV.

scholarly journals Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™ - Antigen-detecting point-of-care device for SARS-CoV-2

2021 ◽  
Author(s):  
Lisa Johanna Krüger ◽  
Julian A.F. Klein ◽  
Frank Tobian ◽  
Mary Gaeddert ◽  
Federica Lainati ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Scale Up ◽  
Cross Reactivity ◽  
Ease Of Use ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Clinical Sensitivity ◽  
Lateral Flow Assays

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

Development and application of loop-mediated isothermal amplification label-based nanoparticles lateral flow biosensor for detection of Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Xingyun Wang ◽  
Yi Wang ◽  
Weiwei Jiao ◽  
Guirong Wang ◽  
Yacui Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Technique ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Morbidity ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Abstract Tuberculosis is a serious disease with high morbidity and mortality, thus rapid and cost-effective diagnostic test for Mycobacterium tuberculosis (MTB) is urgently needed. Here, a novel detection diagnostic technique, termed as loop-mediated isothermal amplification label-based nanoparticles with lateral flow biosensor (LAMP-LFB), was developed and evaluated for rapid, reliable and objective detection of MTB. Two sets of primers, which targeted IS 6110 and IS 1081 sequences of MTB, were simultaneously designed for establishment of LAMP-LFB assay. The optimal reaction conditions of MTB-LAMP-LFB assay confirmed were 66ºC for only 50min. The analytical sensitivity of MTB-LAMP-LFB is 10fg of genomic templates in pure culture, and the detection results obtained from LFB was in conformity with agarose gel electrophoresis. No cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains (NTM) was obtained. A total of 158 clinical samples were collected from presumptive 158 TB patients, were used for evaluating the feasibility of MTB-LAMP-LFB assay. Among 98 TB patients diagnosed with composite reference standard, the positive rate for MTB detection using liquid culture, Xpert MTB/RIF and LAMP-LFB were 40.0% (39/98), 50.0% (48/98), and 86.7% (85/98), respectively. Among 39 culture confirmed samples, 84.6% (33/39) cases were Xpert MTB/RIF-positive and 92.3% (36/39) were LAMP-LFB-positive. For the 59 clinically diagnosed TB cases 25.4% (15/59) and 83.0% (49/59) were Xpert MTB/RIF-positive and LAMP-LFB positive, respectively. Therefore, MTB-LAMP-LFB assay is a simple, reliable, and sensitive method for MTB detection and maybe prospective in early diagnosis of MTB.

scholarly journals Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2

Infection ◽  
2021 ◽  
Author(s):  
Lisa J. Krüger ◽  
Julian A. F. Klein ◽  
Frank Tobian ◽  
Mary Gaeddert ◽  
Federica Lainati ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Scale Up ◽  
Cross Reactivity ◽  
Ease Of Use ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Clinical Sensitivity ◽  
Lateral Flow Assays

Abstract Purpose Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Methods This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2–87.5%). Specificity was 99.3% (CI 98.3–99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2–97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2–56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling. Trial registration number and registration date DRKS00021220 and 01.04.2020

scholarly journals A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258819
Author(s):  
Benjamin D. Grant ◽  
Caitlin E. Anderson ◽  
Luis F. Alonzo ◽  
Spencer H. Garing ◽  
John R. Williford ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Lateral Flow ◽  
World Health ◽  
Analytical Sensitivity ◽  
Rapid Diagnostics ◽  
Middle Income ◽  
Efficient Detection ◽  
Gamma Irradiated ◽  
Health Organization

Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.

scholarly journals Lateral Flow Assay with Near-Infrared Dye for Multiplex Detection

2013 ◽  
Vol 59 (4) ◽  
pp. 641-648 ◽  
Author(s):  
Christina Swanson ◽  
Annalisa D'Andrea
Keyword(s):  
Near Infrared ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Test Strip ◽  
Lateral Flow ◽  
Diagnostic Tools ◽  
High Signal ◽  
Analytical Sensitivity ◽  
Robust Detection ◽  
Lateral Flow Assays

BACKGROUND Lateral flow assays (LFAs) are popular point-of-care diagnostic tools because they are rapid and easy to use. Nevertheless, they often lack analytical sensitivity and quantitative output and may be difficult to multiplex, limiting their usefulness in biomarker measurement. As a proof-of-concept study, we detail the design of a quantitative, multiplex LFA with readily available near-infrared (NIR) detection to improve analytical sensitivity. METHODS NIR dye was conjugated to selected antibodies and incorporated into LFAs. We used singleplex, optimized NIR-LFAs to measure interleukin (IL)-6 from 0 to 200 pg/mL and developed duplex assays to simultaneously measure IL-6 from 0 to 100 pg/mL (0 to 4.5 pmol/L) and C-reactive protein (CRP) from 50 to 2500 ng/mL (0.4 to 20 nmol/L) on a single test strip. Assays were tested on 60 different spiked samples and compared to ELISA results. RESULTS NIR-LFAs detected IL-6 in a 10% plasma matrix with a limit of detection of 4 pg/mL (182 fmol/L) and a CV <7%. Duplex NIR-LFAs quantitatively measured IL-6 and CRP concentrations simultaneously. Values strongly correlated to ELISA measurements, with R2 values of 0.9825 and 0.9711 for IL-6 and CRP, respectively. CONCLUSIONS NIR-LFAs exhibit quantitative measurement at pg/mL concentrations owing to a high signal-to-BACKGROUND ratio and robust detection antibody clearance through the test strip. Moreover, NIR-LFAs are able to detect molecules present at vastly different concentrations in multiplex format and compare favorably to ELISAs. LFAs with direct NIR detection may be a valuable tool for biomarker evaluation in the point-of-care setting.

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM).The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated at a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM). The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

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