Dataset Information

Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

ABSTRACT: Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

SUBMITTER: Jung JK

PROVIDER: S-EPMC11244977 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Jung Jaeyoung K JK Dreyer Kathleen S KS Dray Kate E KE Muldoon Joseph J JJ George Jithin J Shirman Sasha S Cabezas Maria D MD D'Aquino Anne E AE Verosloff Matthew S MS Seki Kosuke K Rybnicky Grant A GA Alam Khalid K KK Bagheri Neda N Jewett Michael C MC Leonard Joshua N JN Mangan Niall M NM Lucks Julius B JB

bioRxiv : the preprint server for biology 20240703

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test u ...[more]

PMID: 39005318

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Project description:Endoscopic technique is often applied for the diagnosis of diseases affecting internal organs and image-guidance of surgical procedures. Although the endoscope has become an indispensable tool in the clinic, its utility has been limited to medical offices or operating rooms because of the large size of its ancillary devices. In addition, the basic design and imaging capability of the system have remained relatively unchanged for decades.The objective of this study was to develop a smartphone-based endoscope system capable of advanced endoscopic functionalities in a compact size and at an affordable cost and to demonstrate its feasibility of point-of-care through human subject imaging.We developed and designed to set up a smartphone-based endoscope system, incorporating a portable light source, relay-lens, custom adapter, and homebuilt Android app. We attached three different types of existing rigid or flexible endoscopic probes to our system and captured the endoscopic images using the homebuilt app. Both smartphone-based endoscope system and commercialized clinical endoscope system were utilized to compare the imaging quality and performance. Connecting the head-mounted display (HMD) wirelessly, the smartphone-based endoscope system could superimpose an endoscopic image to real-world view.A total of 15 volunteers who were accepted into our study were captured using our smartphone-based endoscope system, as well as the commercialized clinical endoscope system. It was found that the imaging performance of our device had acceptable quality compared with that of the conventional endoscope system in the clinical setting. In addition, images captured from the HMD used in the smartphone-based endoscope system improved eye-hand coordination between the manipulating site and the smartphone screen, which in turn reduced spatial disorientation.The performance of our endoscope system was evaluated against a commercial system in routine otolaryngology examinations. We also demonstrated and evaluated the feasibility of conducting endoscopic procedures through a custom HMD.

Dataset Information

Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Publications

Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

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