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 Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32

2022 ◽  
Vol 16 (1) ◽  
pp. e0010112
Author(s):  
Sirawit Jirawannaporn ◽  
Umaporn Limothai ◽  
Sasipha Tachaboon ◽  
Janejira Dinhuzen ◽  
Patcharakorn Kiatamornrak ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Performance ◽  
Point Of Care ◽  
Limited Resource ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Detection Assay ◽  
Short Palindromic Repeat ◽  
Flow Detection ◽  
Point Of Care Detection

Background One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. Methodology/Principal findings A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4–6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. Conclusions/Significance The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

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.

Diagnostic tests for detecting Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and pharyngeal specimens

2021 ◽  
Author(s):  
Paul C. Adamson ◽  
Jeffrey D. Klausner
Keyword(s):  
Chlamydia Trachomatis ◽  
Neisseria Gonorrhoeae ◽  
Bacterial Infections ◽  
Point Of Care ◽  
High Sensitivity ◽  
Population Level ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Point Of Care Test ◽  
Control And Prevention

Chlamydia trachomatis and Neisseria gonorrhoeae are two of the most often reported bacterial infections in the United States. The rectum and oropharynx are important anatomic sites of infection and can contribute to ongoing transmission. Nucleic acid amplification tests (NAATs) are the mainstays for the detection of C. trachomatis and N. gonorrhoeae infections owing to their high sensitivity and specificity. Several NAATs have been evaluated for testing in rectal and pharyngeal infections. A few assays recently received clearance by the Food and Drug Administration, including one point-of-care test. Those assays can be used for testing in symptomatic individuals, as well as for asymptomatic screening in certain patient populations. Routine screening for C. trachomatis in pharyngeal specimens is not recommended by the Centers for Disease Control and Prevention, though is often performed due to the use of multiplex assays. While expanding the types of settings for screening and using self-collected rectal and pharyngeal specimens can help to increase access and uptake of testing, additional research is needed to determine the potential benefits and costs associated with increased screening for rectal and pharyngeal C. trachomatis and N. gonorrhoeae infections on a population level.

scholarly journals Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study

2020 ◽  
Author(s):  
Azeem Mehmood Butt ◽  
Shafiqa Siddique ◽  
Xiaoping An ◽  
Yigang Tong
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification ◽  
Qualitative Detection ◽  
Detection Assay ◽  
Testing Protocols ◽  
Positive Results

AbstractSevere acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) and 25 non-COVID-19 cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive results in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.

scholarly journals Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

2020 ◽  
Author(s):  
A. Ganguli ◽  
A. Mostafa ◽  
J. Berger ◽  
M. Aydin ◽  
F. Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Rna Extraction ◽  
Clinical Diagnostics ◽  
Isothermal Amplification ◽  
Sample Collection ◽  
Rt Pcr ◽  
Point Of Use ◽  
Viral Transport Medium

AbstractThe COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/μL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/μL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

scholarly journals A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care

2020 ◽  
Author(s):  
Diem Hong Tran ◽  
Hoang Quoc Cuong ◽  
Hau Thi Tran ◽  
Uyen Phuong Le ◽  
Hoang Dang Khoa Do ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
Nucleic Acid Amplification ◽  
Gold Standard Method ◽  
Synthetic Dna ◽  
Isothermal Nucleic Acid Amplification ◽  
Freeze Dried ◽  
Test Kit

ABSTRACTThe COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-to-use kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

scholarly journals Rapid Multiplexed Detection on Lateral-Flow Devices Using a Laser Direct-Write Technique

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 97 ◽  
Author(s):  
Peijun He ◽  
Ioannis Katis ◽  
Robert Eason ◽  
Collin Sones
Keyword(s):  
Bacterial Infections ◽  
Point Of Care ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Direct Write ◽  
Multiplexed Detection ◽  
Amyloid A ◽  
Flow Devices ◽  
Laser Direct Write

Paper-based lateral flow devices (LFDs) are regarded as ideal low-cost diagnostic solutions for point-of-care (POC) scenarios that allow rapid detection of a single analyte within a fluidic sample, and have been in common use for a decade. In recent years, there has been an increasing need for rapid and simultaneous detection of multiple analytes present within a single sample and to facilitate this, we report here a novel solution—detection using a multi-path LFD created via the precise partitioning of the single flow-path of a standard LFD using our previously reported laser direct-write (LDW) technique. The multiple flow-paths allow the simultaneous detection of the different analytes individually within each of the parallel channels without any cross-reactivity. The appearance of coloured test lines in individual channels indicates the presence of the different analytes within a sample. We successfully present the use of a LDW-patterned multi-path LFD for multiplexed detection of a biomarker panel comprising C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), used for the diagnosis of bacterial infections. Overall, we demonstrate the use of our LDW technique in the creation of a novel LFD that enables multiplexed detection of two inflammation markers within a single LFD providing a detection protocol that is comparatively more efficient than the standard sequential multiplexing procedure.

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.

scholarly journals Comparison of FoodChek E. coli Test and AOAC Research Institute Performance Tested Method 010603 for Detection of E. coli O157 in Raw Ground Beef

2010 ◽  
Vol 93 (3) ◽  
pp. 922-927 ◽  
Author(s):  
Richard J Flanagan ◽  
Gabriela Martinez
Keyword(s):  
Magnetic Nanoparticle ◽  
Raw Materials ◽  
Detection System ◽  
Detection Threshold ◽  
Reference Method ◽  
Superparamagnetic Nanoparticles ◽  
E Coli ◽  
Detection Assay ◽  
Highly Sensitive ◽  
Research Institute

Abstract The FoodChek E. coli O157 assay [AOAC Research Institute (RI) Performance Tested Method (PTM) 060902] is a rapid detection system that incorporates the use of antibody-coated superparamagnetic nanoparticles in a lateral flow immunoassay format. The system comprises a commercially available enrichment medium, a magnetic nanoparticle immunoassay, and an automated reader for detection. Assay detection threshold is improved relative to traditional immunoassays through use of a magnetic nanoparticle label and a highly sensitive magnetic particle detector. FoodChek E. coli O157 is a reintroduction of the previously certified AOAC PTM 010603. The original assay was evaluated and approved in internal and independent laboratory studies. Vacci-Test Corp. has contracted with the original supplier of the PTM to remanufacture the test under identical conditions and with identical raw materials. This report is intended to show that FoodChek E. coli is identical in performance to the previously approved PTM. The results showed no difference for the parameters evaluated. Three kit lots along with three lots of media and media supplement were compared in lot-to-lot and stability testing. The results indicated no difference in performance across the three lots. The results showed sensitivity of >99 and a specificity rate of >98 for the FoodChek method and a significantly higher sensitivity than the reference method.

Noncompetitive Immunoassay Detection System for Haptens on the Basis of Antimetatype Antibodies

2015 ◽  
Vol 61 (4) ◽  
pp. 627-635 ◽  
Author(s):  
Kazuya Omi ◽  
Tsuyoshi Ando ◽  
Takuya Sakyu ◽  
Takashi Shirakawa ◽  
Yoshiaki Uchida ◽  
...  
Keyword(s):  
Small Molecules ◽  
Limit Of Detection ◽  
Ex Vivo ◽  
Detection System ◽  
Sandwich Elisa ◽  
Microtiter Plate ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D

Abstract BACKGROUND Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS We generated antimetatype monoclonal antibodies against a hapten–antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%–12.6% (intraassay) and 6.2%–21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%–2.3% (intraassay) and 1.9%–3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available 125I RIA. CONCLUSIONS Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

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