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A HiPAD Integrated with rGO/MWCNTs Nano-Circuit Heater for Visual Point-of-Care Testing of SARS-CoV-2.

ABSTRACT: Nowadays, the main obstacle for further miniaturization and integration of nucleic acids point-of-care testing devices is the lack of low-cost and high-performance heating materials for supporting reliable nucleic acids amplification. Herein, reduced graphene oxide hybridized multi-walled carbon nanotubes nano-circuit integrated into an ingenious paper-based heater is developed, which is integrated into a paper-based analytical device (named HiPAD). The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still raging across the world. As a proof of concept, the HiPAD is utilized to visually detect the SARS-CoV-2 N gene using colored loop-mediated isothermal amplification reaction. This HiPAD costing a few dollars has comparable detection performance to traditional nucleic acids amplifier costing thousands of dollars. The detection range is from 25 to 2.5 × 10¹⁰ copies mL^-1 in 45 min. The detection limit of 25 copies mL^-1 is 40 times more sensitive than 1000 copies mL^-1 in conventional real-time PCR instruments. The disposable paper-based chip could also avoid potential secondary transmission of COVID-19 by convenient incineration to guarantee biosafety. The HiPAD or easily expanded M-HiPAD (for multiplex detection) has great potential for pathogen diagnostics in resource-limited settings.

SUBMITTER: Li S

PROVIDER: S-EPMC8250055 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed.ResultsWe developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/μL of saliva within 30 minutes without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples.SignificanceIACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices.

Project description:ObjectivesLow-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing.MethodsWe compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n = 46) and (b) homebrew integrase (IN) (n = 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only.ResultsThe average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both.ConclusionsThe Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings.

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A HiPAD Integrated with rGO/MWCNTs Nano-Circuit Heater for Visual Point-of-Care Testing of SARS-CoV-2.

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