Clustered Regularly Interspaced Short Palindromic Repeats-Mediated Surface-Enhanced Raman Scattering Assay for Multidrug-Resistant Bacteria

Abstract

Antimicrobial resistance and multidrug resistance are slower-moving pandemics than the fast-spreading coronavirus disease 2019; however, they have potential to cause a much greater threat to global health. Here, we report a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated surface-enhanced Raman scattering (SERS) assay for multidrug-resistant (MDR) bacteria. This assay was developed via a synergistic combination of the specific gene-recognition ability of the CRISPR system, superb sensitivity of SERS, and simple separation property of magnetic nanoparticles. This assay detects three multidrug-resistant (MDR) bacteria, species Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae, without purification or gene amplification steps. Furthermore, MDR A. baumannii-infected mice were successfully diagnosed using the assay. Finally, we demonstrate the on-site capture and detection of MDR bacteria through a combination of the three-dimensional nanopillar array swab and CRISPR-mediated SERS assay. This method may prove effective for the accurate diagnosis of MDR bacterial pathogens, thus preventing severe infection by ensuring appropriate antibiotic treatment.

Biosensors and Point‐of‐Care Devices for Bacterial Detection: Rapid Diagnostics Informing Antibiotic Therapy

“…Finally, the qPCR technique can sometimes lead to false positives which often arise from the sample processing and nucleic acid amplification steps. [34] With regards to sandwich-based ELISA methods, they can also detect bacteria in small sample volumes with high reproducibility. These techniques also use reagents which can last for a long time while being radiation-free and producing non-toxic wastes.…”

Direct MYD88L265P gene detection for diffuse large B-cell lymphoma (DLBCL) via a miniaturised CRISPR/dCas9-based sensing chip

“…Many analytical techniques have been combined with CRISPR/Cas systems for DNA/RNA detection, for instance, fluorescence, 30 liquid-gated graphene field-effect transistors, 23 and surface-enhanced Raman scattering (SERS). 31 Here, we developed a CRISPR-mediated electrochemical detection platform based on the ion-channel feature of the PAA membrane. PAA with a hexagonal close-packed arrangement has broad application prospects because of its simple preparation method, low cost, solid structure and stable physical and chemical properties.…”

“…The developed CRISPR-PAA device was employed to directly test the MYD88 L265P mutation genes of clinical patient genomic DNA samples without amplification or purification. 31 Genome-wide DNA was derived from the tissue removed after surgery from patients with lymphoma.…”

Engineered CRISPR-Cas systems for the detection and control of antibiotic-resistant infections

“…They reverse transcribed the ssRNA of the virus into cDNA and successfully used Cas12a to achieve indirect detection of RNA [ 183 ]. Kim et al [ 184 ] studied CRISPR-mediated surface-enhanced Raman spectroscopy (SERS) analysis, which combines CRISPR-Cas9 with SERS-active Au-coated magnetic nanoparticles (Au MNPs), and applied it to detect antimicrobial resistance genes. Based on the inherent SERS spectrum of bacteria and the high sensitivity of SERS, multiple and accurate identifications of bacteria can be realized [ 185 , 186 ].…”

“…This method successfully detected three MDR bacteria, S. aureus , A. baumannii , and K. pneumoniae , and was verified in a mouse infection model. In addition, these scientists applied a 3D nanopillar array swab, thereby omitting unnecessary sample preparation steps to realize on-site identification of MDR bacteria [ 184 ].…”