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 7 publications

(9 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.

View full text Add to dashboard Cite

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.

show abstract

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.

View full text Add to dashboard Cite

show abstract

“…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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

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

Cited by 7 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

Contact Info

Product

Resources

About