A New Method Based on LAMP-CRISPR–Cas12a-Lateral Flow Immunochromatographic Strip for Detection

Abstract

Introduction Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern. Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options. It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains. Methods For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR–Cas12a, and lateral flow immunochromatographic strip (CRISPR–Cas-LAMP-lateral flow strip). The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system. Results The cleavage products are visible to the naked eye on the lateral flow strip. This method is highly sensitive in direct detection of bacteria in samples containing at least 3×10 5 CFU/mL without the need for bacterial culture. Discussion It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method. This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.

A Method for Detecting Five Carbapenemases in Bacteria Based on CRISPR-Cas12a Multiple RPA Rapid Detection Technology

“…The characteristic of this technology is its suitability for both detection of large-scale indoor populations and the screening of small-scale external populations. 19 …”

Establishment and Methodological Evaluation of a Method for Rapid Detection of Helicobacter pylori and Virulence Genes Based on CRISPR-Cas12a

“…For example, crRNA is easily degraded and the storage conditions are relatively strict, requiring it to be placed at −20°C or below. 33 Therefore, storage methods and conditions need to be improved to ensure a longer shelf-life at room temperature for large-scale and extensive clinical use. We consider that not being dependent on instruments will facilitate the universal detection of various pathogens, including H. pylori .…”

Nanoparticle-Based Visual Detection of Amplified DNA for Diagnosis of Hepatitis C Virus

“…To detect captured DNA, signal detection steps, including fluorescent scanning [ 27 ] or visual RT-LAMP-CRISPR detection under a blue light illuminator, are required [ 28 ]. However, STAT-DNA does not require a lateral flow dipstick, membrane paper, a dispensing system for line dispensing, protein blockers, stabilizers, immobilization of the capture reagent in the membrane [ 29 , 30 ], or a detection procedure, such as a hybridization step with heating [ 26 ]. In our previous studies, several factors affected the migration of particles in the SPIN-DNA [ 17 ] and PASS-DNA [ 9 ] systems, such as Sepharose and intercalating dye concentration, centrifugal force, and Cy3 labeling.…”