Abstract:Field-deployable
detectors of disease biomarkers provide a simple
and fast analysis of clinical specimens. However, most of the existing
field-deployable diagnostics have poor sensitivity and are not suitable
for the detection of biomarkers with low abundance. Herein, we report
a highly sensitive and rapid colorimetric readout paper-based assay
for pathogen detection by integrating the unique collateral activity
of a Cas12a-activated universal field-deployable detector (CUFD).
The collateral effect of Cas12a r… Show more
“… 25 Hence, it is applicable for on-site analysis of nucleic acids in samples with greatly enhanced detection efficiency and convenience. 26 Figure 3 Research design and amplification results: some target genes were successfully amplified by PCR ( A ) and LAMP ( B ), and the corresponding PCR and LAMP bands were obtained by agarose gel electrophoresis. For PCR ( A ), 1–6 were positive samples, and 7 and 8 were negative samples.…”
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.
“… 25 Hence, it is applicable for on-site analysis of nucleic acids in samples with greatly enhanced detection efficiency and convenience. 26 Figure 3 Research design and amplification results: some target genes were successfully amplified by PCR ( A ) and LAMP ( B ), and the corresponding PCR and LAMP bands were obtained by agarose gel electrophoresis. For PCR ( A ), 1–6 were positive samples, and 7 and 8 were negative samples.…”
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.
“…Once developed, the lateral flow-based SHERLOCK and DETECTR assays have found wide applications to the diagnosis of infectious diseases caused by Zika virus, SARS-CoV-2 and pathogenic bacteria, such as P. aeruginosa , S. dysenteriae , etc . 160–163…”
Section: Analytical Assay Development Using the Crispr–cas Toolboxmentioning
“…[32,78] Furthermore, CRISPR/Cas12a-mediated LFAs based on AuNPsbased colorimetric readout have been widely established for the detection of multiple analytes. [66,67,73,75,76,[79][80][81][82][83] By employing the programmable CRISPR/Cas12a systems, AuNPs probe, and LFA, Cheng et al established a novel colorimetric code system for the analysis of telomeric repeat DNA and internal control in telomeric repeat amplification protocol (TRAP) products, facilitating a fast determination of the telomerase activity and an easy identification of false-negative (FN) results via naked [32] Copyright 2020, American Chemical Society. B) Schematic illustration of virus detection based on CRISPR/dCas9.…”
Infectious pathogens cause severe human illnesses and great deaths per year worldwide. Rapid, sensitive, and accurate detection of pathogens is of great importance for preventing infectious diseases caused by pathogens and optimizing medical healthcare systems. Inspired by a microbial defense system (i.e., CRISPR/ CRISPR-associated proteins (Cas) system, an adaptive immune system for protecting microorganisms from being attacked by invading species), a great many new biosensors have been successfully developed and widely applied in the detection of infectious viruses and pathogenic bacteria. Moreover, advanced nanotechnologies have also been integrated into these biosensors to improve their detection stability, sensitivity, and accuracy. In this review, the recent advance in CRISPR/Cas systems-based nano/biosensors and their applications in the detection of infectious viruses and pathogenic bacteria are comprehensively reviewed. First of all, the categories and working principles of CRISPR/Cas systems for establishing the nano/biosensors are simply introduced. Then, the design and construction of CRISPR/Cas systems-based nano/biosensors are comprehensively discussed. In the end, attentions are focused on the applications of CRISPR/Cas systems-based nano/biosensors in the detection of infectious viruses and pathogenic bacteria. Impressively, the remaining opportunities and challenges for the further design and development of CRISPR/Cas system-based nano/biosensors and their promising applications are proposed.
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