Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus

2020 ◽  
Author(s):  
Thomas R. Shelite ◽  
Nathen E. Bopp ◽  
Abelardo Moncayo ◽  
Erin S. Reynolds ◽  
Saravanan Thangamani ◽  
...  
Keyword(s):  
Diagnostic Test ◽  
Point Of Care ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification

scholarly journals Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

2020 ◽  
Author(s):  
Thomas R Shelite ◽  
Ashanti C Uscanga-Palomeque ◽  
Alejandro Castellanos ◽  
Peter C Melby ◽  
Bruno L Travi
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
The United States ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Reference Test ◽  
Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

scholarly journals Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

Foods ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 27 ◽  
Author(s):  
Jiali Li ◽  
Biao Ma ◽  
Jiehong Fang ◽  
Antong Zhi ◽  
Erjing Chen ◽  
...  
Keyword(s):  
Test Line ◽  
Point Of Care ◽  
Limited Resource ◽  
Test Strip ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Lateral Flow Immunoassay ◽  
Quantitative Detection ◽  
Control Line ◽  
Test Strip Reader

Salmonella can cause serious foodborne diseases. We have developed a lateral flow immunoassay combined with recombinase polymerase amplification (LFD-RPA) for detection of Salmonella in food. The conserved fragment (fimY) was selected as the target gene. Under an optimal condition (37 °C, 10 min), the sensitivity was 12 colony-forming units (CFU)/mL in a pure culture. Testing with 16 non-Salmonella strains as controls revealed that LFD-RPA was specific to the fimY gene of Salmonella. The established assay could detect Salmonella at concentrations as low as 1.29 × 102 CFU/mL in artificially contaminated samples. This detection was at a slightly higher level than that for a pure bacterial culture. Combined with the test strip reader, the LFD-RPA is a feasible method for quantitative detection of Salmonella based on the test line intensity, which was the ratio for the test line and control line of the reflected light. The method could be a potential point-of-care test in limited resource areas and provides a new approach and technical support for the diagnosis of food safety.

A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Dan Liu ◽  
Haicong Shen ◽  
Yuqian Zhang ◽  
Danyu Shen ◽  
Mingyang Zhu ◽  
...  
Keyword(s):  
Point Of Care ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Point Of Care Testing ◽  
Resource Limited ◽  
Medical Crisis

The COVID-19 pandemic, caused by SARS-CoV-2, currently poses an urgent global medical crisis for which there remains a lack of affordable point-of-care testing (POCT). In particular, resource-limited areas need simple...

scholarly journals Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

2020 ◽  
Author(s):  
Thomas R Shelite ◽  
Ashanti C Uscanga-Palomeque ◽  
Alejandro Castellanos ◽  
Peter C Melby ◽  
Bruno L Travi
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
The United States ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Reference Test ◽  
Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

scholarly journals Rapid Detection of Burkholderia pseudomallei with a Lateral Flow Recombinase Polymerase Amplification Assay

2019 ◽  
Author(s):  
Yao Peng ◽  
Zheng Xiao ◽  
Biao Kan ◽  
Wei Li ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Burkholderia Pseudomallei ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Spiked Soil ◽  
Lateral Flow Dipstick ◽  
Life Threatening ◽  
Amplification Assay

AbstractMelioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection benefits greatly to implement effective treatment timely, which is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B.pseudomallei. A set of probe and primers targeting orf2 gene of B. pseudomallei were generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LoD) as low as 20 femtogram (ca. 25.6 copies) of B. pseudomallei genomic DNA. The assay is highly specific as no cross amplification was observed with 35 non-B. pseudomallei pathogens. Isolates (N=19) from patients of Hainan province of China were retrospectively confirmed by the newly developed method. LoD for B. pseudomallei spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. Sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR, however, the LF-RPA assay exhibited a better tolerant to inhibitors in blood than the later. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

scholarly journals Evaluation and comparison of recombinase polymerase amplification coupled with lateral-flow bioassay for Escherichia coli O157:H7 detection using diifeerent genes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alka Rani ◽  
Vivek B. Ravindran ◽  
Aravind Surapaneni ◽  
Esmaeil Shahsavari ◽  
Nagalakshmi Haleyur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Professional Training ◽  
Gene Selection ◽  
Point Of Care ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Gene Target ◽  
Microbial Detection ◽  
E Coli ◽  
Simple Detection

AbstractShiga toxin-producing Escherichia coli serotype O157:H7 is a food and waterborne zoonotic pathogen causing gastroenteritis in humans. Rapid and simple detection in water and food is imperative to control its spread. However, traditional microbial detection approaches are time-consuming, expensive and complex to operate at the point-of-care without professional training. We present a rapid, simple, sensitive, specific and portable method for detection of E. coli O157:H7 in drinking water, apple juice and milk. We evaluated the effect of gene selection in detecting E. coli O157:H7 using recombinase polymerase amplification coupled with a lateral flow assay using rfbE, fliC and stx gene targets. As low as 100 ag and 1 fg DNA, 4–5 CFU/mL and 101 CFU/mL of E. coli O157:H7 was detected using the stx and rfbE gene targets respectively with 100% specificity, whilst the detection limit was 10 fg DNA and 102 CFU/mL for the fliC gene target, with 72.8% specificity. The RPA-LFA can be completed within 8 min at temperatures between 37 and 42 °C with reduced handling and simple equipment requirements. The test threshold amplification of the target was achieved in 5–30 min of incubation. In conclusion, RPA-LFA represents a potential rapid and effective alternative to conventional methods for the monitoring of E. coli O157:H7 in food and water.

Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

2021 ◽  
Author(s):  
David Cate ◽  
Helen Hsieh ◽  
Veronika Glukhova ◽  
Joshua D Bishop ◽  
H Gleda Hermansky ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Point Of Care ◽  
Lateral Flow ◽  
Screening Methods ◽  
Antibody Screening ◽  
Liquid Handling ◽  
Large Numbers ◽  
Accurate Performance ◽  
Further Development ◽  
Assay Conditions

<p></p><p>The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow immunoassay (LFA) tests in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.</p><p></p>

Gold-Aptamer-Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

2019 ◽  
Author(s):  
Veeren Chauhan ◽  
Mohamed M Elsutohy ◽  
C Patrick McClure ◽  
Will Irving ◽  
Neil Roddis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
In Silico ◽  
Point Of Care ◽  
Lateral Flow ◽  
Nucleic Acid Sequence ◽  
In Silico Screening ◽  
Lateral Flow Assays ◽  
Life Threatening ◽  
Software And Hardware ◽  
Nucleic Acid Sequences

<p>Enteroviruses are a ubiquitous mammalian pathogen that can produce mild to life-threatening disease. Bearing this in mind, we have developed a rapid, accurate and economical point-of-care biosensor that can detect a nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and aptamers to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral RNA sequence (23 bases), which was identified through in silico screening. Aptamers were designed to demonstrate specific complementarity towards the target enteroviral RNA to produce aggregated gold-aptamer nanoconstructs. Conserved target enteroviral nucleic acid sequence (≥ 1x10<sup>-7</sup> M, ≥1.4×10<sup>-14</sup> g/mL), initiates gold-aptamer-nanoconstructs disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow-assays that utilise gold-aptamer-nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (< 60 s) and could be interpreted with a bespoke software and hardware electronic interface. We anticipate our methodology will translate in-silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave-the-way forward in the clinical evaluation of disease and complement existing strategies at overcoming antimicrobial resistance.</p>

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