Automated Microfluidic Nucleic Acid Detection Platform-Integrated RPA-T7-Cas13a for Pathogen Diagnosis

Abstract

There is a growing urgent need for point-of-care testing (POCT) devices that integrate sample pretreatment and nucleic acid detection in a rapid, economical, and non-laborintensive way. Here, we have developed an automated, portable nucleic acid detection system employing microfluidic chips integrating rotary valve-assisted sample pretreatment and recombinase polymerase amplification (RPA)-T7-Cas13a into one-step nucleic acid detection. The RPA and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a were integrated into a single-chamber reaction. As a validation model, we used this method to detect Group B streptococci (GBS) DNA and achieved a detection sensitivity of 8 copies/reaction, which is 6 times more sensitive than gold-standard polymerase chain reactions (PCRs). Dual specific recognition of RPA with CRISPR/Cas13a makes our method ultraspecific, with correct detection of Group B streptococci from 8 kinds of pathogenic bacteria. For the 16 positive and 24 negative clinical GBS samples, our assay achieved 100% accuracy compared to the PCR technique. The whole procedure can be automatically completed within 30 min, providing a more robust, sensitive, and accurate molecular diagnostic tool for POCT.

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

“…Meanwhile, CRISPR-Cas13a was also used to identify a number of bacterial strains, including Aspergillus fumigatus ( Li et al, 2022 ), Staphylococcus aureus ( Zhou et al, 2020 ), Salmonella spp. ( An et al, 2021 ), Neisseria gonorrhoeae ( Allan-Blitz et al, 2023 ) and Group B Streptococci (GBS; Li Z. et al, 2023 ). Moreover, the CRISPR-Cas13a technology was also utilized to identify multiple genes, such as toxin genes in Clostridioides difficile ( Jiang et al, 2023 ), the mecA and clfA genes in methicillin-resistant S. aureus (MRSA; Liu et al, 2023 ), the blaKPC gene in Enterobacterales ( Liang et al, 2023 ), the lcrV gene in Yersinia pestis ( Schultzhaus et al, 2021 ).…”

Exonuclease III/Cas12a Cascade Amplification Strategy and Smartphone-Based Portable Fluorescence Detector to Repurpose the Commercial AFP Strip for the POCT of Multiple RNAs

“…The RT-qPCR not only relies on sophisticated instrumentations and professional laboratories but also requires people coming to specific locations for sample collection, which may increase the risk of cross infection. Although there are excellent ideas with potential in creating NA-POCT devices, they often take long period of time to be transformed into commercially viable products. − Thus, these advanced ideas may not produce immediate effects at the time of need. In order to shorten the idea-to-market time, a strategy that repurposes existing POCT devices for NAT has been proposed, as this strategy reduces the strenuous process for original design, optimization, and manufacture.…”

Integrated Microwell Array-Based Microfluidic Chip with a Hand-Held Smartphone-Controlled Device for Nucleic Acid Detection

“…In the past 30 years, microfluidic chips have led to significant advances in chemistry, biology, and medicine. − As many structures and functions can be integrated into miniaturized devices, microfluidic chips have been developed as important platforms for biochemical reactions, − especially in the point-of-care testing (POCT) applications. − …”