Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Biyani, Radhika; Sharma, Kirti Rani; Kojima, Kenji; Biyani, Madhu; Sharma, Vishnu L.; Kumawat, Tarun; Juma, Kevin Maafu; Yanagihara, Itaru; Fujiwara, Shinsuke; Kodama, Eiichi; Takamura, Yuzuru; Takagi, Masahiro; Yasukawa, Kiyoshi; Biyani, Manish

doi:10.1038/s41598-021-95411-x

Cited by 8 publications

(9 citation statements)

References 34 publications

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“…According to the 3σ rule, the determined limit of detection (LOD) of the portable BDCHA-based biosensor was 291.48 aM, showing good sensitivity of the proposed method. The proposed sensor outperformed recently reported methods − (Table S4) in detection time and sensitivity.…”

Section: Resultsmentioning

confidence: 56%

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a “Booster” for SARS-CoV-2 Point-of-Care Testing

Chen,

Yang,

et al. 2023

Anal. Chem.

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Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). However, the low efficiency of target recycle amplification and the steric hindrance at the electrode interface limit their sensing performance. Herein, we propose a base-stacking-driven catalytic hairpin assembly (BDCHA), a nucleic acid amplification reaction strategy, for POCT. The introduction of the base-stacking effect in this strategy increases the thermodynamic stability of the product, thereby effectively improving the recycling efficiency. Also, it enables the interface-mediated hybridization to maintain stability with even fewer bases in the reaction-binding domain, hence minimizing DNA secondary structure formation or intertwining at the electrode surface and ameliorating the steric hindrance limitation. The introduced base-stacking effect makes the electrode serve as a “booster” by integrating the advantages of homogeneous and heterogeneous reactions, giving BDCHA an increased reaction rate of about 20-fold, compared to the conventional catalytic hairpin assembly. As a proof of concept, our BDCHA was applied in constructing a portable E-DNA biosensor for the detection of a SARS-CoV-2 N gene sequence fragment. A simple 30 min one-pot incubation is required, and the results can be readily read on a smartphone, making it portable and user-friendly for POCT.

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Section: Resultsmentioning

confidence: 56%

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a “Booster” for SARS-CoV-2 Point-of-Care Testing

Chen,

Yang,

et al. 2023

Anal. Chem.

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“…In addition, to increase the efficiency of the Nanotrap microbiome particles, we used a cellulose–acetate filter during sampling to undertake cold sterilization of the wastewater medium for the absolute elimination of yeasts and molds, but not viruses [ 45 ]. Next, the direct amplification of viral lysates from Nanotrap microbiome particles utilizing RICCA (RNA Isothermal Co-assisted and Coupled Amplification), an ultra-sensitive and ultrarapid, one-pot isothermal nucleic acid amplification, allowed us to achieve lab-free robustness [ 46 ]. Furthermore, we also report that following a qualitative analysis of SARS-CoV-2 in wastewater by RICCA, lab-free quantification of viral RNA can be performed by applying viral lysates from Nanotrap microbiome particles on PicoGene PCR1100 (Nippon Sheet Glass, Tokyo, Japan), an improved alternative microfluidic mobile qPCR [ 47 ].…”

Section: Introductionmentioning

confidence: 99%

Real-Time On-Site Monitoring of Viruses in Wastewater Using Nanotrap® Particles and RICCA Technologies

Sharma,

Takamura,

Biyani

et al. 2024

Biosensors

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Wastewater-based epidemiology (WBE) is an effective and efficient tool for the early detection of infectious disease outbreaks in a community. However, currently available methods are laborious, costly, and time-consuming due to the low concentration of viruses and the presence of matrix chemicals in wastewater that may interfere with molecular analyses. In the present study, we designed a highly sensitive “Quick Poop (wastewater with fecal waste) Sensor” (termed, QPsor) using a joint approach of Nanotrap microbiome particles and RICCA (RNA Isothermal Co-Assisted and Coupled Amplification). Using QPsor, the WBE study showed a strong correlation with standard PEG concentrations and the qPCR technique. Using a closed format for a paper-based lateral flow assay, we were able to demonstrate the potential of our assay as a real-time, point-of-care test by detecting the heat-inactivated SARS-CoV-2 virus in wastewater at concentrations of 100 copies/mL and within one hour. As a proof-of-concept demonstration, we analyzed the presence of viral RNA of the SARS-CoV-2 virus and PMMoV in raw wastewater samples from wastewater treatment plants on-site and within 60 min. The results show that the QPsor method can be an effective tool for disease outbreak detection by combining an AI-enabled case detection model with real-time on-site viral RNA extraction and amplification, especially in the absence of intensive clinical laboratory facilities. The lab-free, lab-quality test capabilities of QPsor for viral prevalence and transmission in the community can contribute to the efficient management of pandemic situations.

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“…Finally, RT–RPA combined with DNAzyme provided a detection limit of 10 copies/μL within 1 h [ 19 ] or a FAM and biotin-labeled RT–RPA product, 5 copies/μL, detected within 25 min [ 20 ]. In another approach, RT and RPA reactions were simultaneously performed for the analysis of 2.8 copies/μL within 15–30 min [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection

et al. 2022

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Since the onset of the SARS-CoV-2 pandemic, several COVID-19 detection methods, both commercially available and in the lab, have been developed using different biomolecules as analytes and different detection and sampling methods with high analytical performance. Developing novel COVID-19 detection assays is an exciting research field, as rapid accurate diagnosis is a valuable tool to control the current pandemic, and also because the acquired knowledge can be deployed for facing future infectious outbreaks. We here developed a novel gold-nanoparticle-based nucleic acid lateral flow assay for the rapid, visual, and quantitative detection of SARS-CoV-2. Our method was based on the use of a DNA internal standard (competitor) for quantification and involved RT-PCR, the hybridization of biotinylated PCR products to specific oligonucleotide probes, and detection with a dual lateral flow assay using gold nanoparticles conjugated to an anti-biotin antibody as reporters. The developed test allowed for rapid detection by the naked eye and the simultaneous quantification of SARS-CoV-2 in nasopharyngeal swabs with high specificity, detectability, and repeatability. This novel molecular strip test for COVID-19 detection represents a simple, cost-effective, and accurate rapid test that is very promising to be used as a future diagnostic tool.

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Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Cited by 8 publications

References 34 publications

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a “Booster” for SARS-CoV-2 Point-of-Care Testing

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a “Booster” for SARS-CoV-2 Point-of-Care Testing

Real-Time On-Site Monitoring of Viruses in Wastewater Using Nanotrap® Particles and RICCA Technologies

A Molecular Lateral Flow Assay for SARS-CoV-2 Quantitative Detection

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