Loop-Mediated Isothermal Amplification-Integrated CRISPR Methods for Infectious Disease Diagnosis at Point of Care

Abstract

Infectious diseases continue to pose an imminent threat to global public health, leading to high numbers of deaths every year and disproportionately impacting developing countries where access to healthcare is limited. Biological, environmental, and social phenomena, including climate change, globalization, increased population density, and social inequity, contribute to the emergence of novel communicable diseases. Rapid and accurate diagnoses of infectious diseases are essential to preventing the transmission of infectious diseases. Although some commonly used diagnostic technologies provide highly sensitive and specific measurements, limitations including the requirement for complex equipment/infrastructure and refrigeration, the need for trained personnel, long sample processing times, and high cost remain unresolved. To ensure global access to affordable diagnostic methods, loop-mediated isothermal amplification (LAMP) integrated clustered regularly interspaced short palindromic repeat (CRISPR) based pathogen detection has emerged as a promising technology. Here, LAMP-integrated CRISPR-based nucleic acid detection methods are discussed in point-of-care (PoC) pathogen detection platforms, and current limitations and future directions are also identified.

Figure 1

Overview of a conventional LAMP-integrated CRISPR workflow for PoC platforms. (A) The LAMP-integrated CRISPR-based diagnostic mechanism consists of (i) sample collection; (ii) sample preparation; (iii) loop-mediated isothermal amplification at 60–65 °C; (iv) CRISPR-based detection, leveraging the collateral cleavage activity of Cas12 and Cas13 to cleave nontarget nucleic acid molecules upon target recognition; and (v) interpretation of results using fluorescence or LFA. (B) Single-tube platforms are used to reduce contamination, simplify the process for untrained users, and streamline testing. Common strategies to develop single-tube platforms are as follows: (i) mineral oil is used to prevent LAMP and CRISPR reagents from mixing, (ii) CRISPR reagents are placed on the lid of the tube, and (iii) thermostable Cas enzymes and/or lyophilized reagents are used. (C) Future prospects to enhance the functionality of LAMP-integrated CRISPR-based PoC platforms: (i) multiplex testing capabilities, (ii) the integration of microfluidics, (iii) miniaturization of devices, and (iv) digitalization of platforms. Some of the elements in Figure 1 were designed using resources from freepik.com and flaticon.com.

Figure 2

ITP-enhanced CRISPR detection of SARS-CoV-2 in nasopharyngeal swab samples. (A) ITP-CRISPR workflow for SARS-CoV-2 detection. (B) Stepwise schematic of incubation, RNA extraction, RT-LAMP, and ITP-CRISPR detection phases. (C) Nucleic acid extraction from a nasopharyngeal swab sample on chip. (D) RT-PCR of ITP-extracted nucleic acid samples obtained from clinical nasopharyngeal samples. D0–D3 Covid-19 samples are used as negative controls. D1–D3 Covid-19 samples are 1:10 serial dilutions of the D0 Covid-19 sample. (E) Fluorescence signals obtained for N, E, and RNase P gene amplification products following RT-LAMP. (F) Analytical LOD for ITP-CRISPR detection. Adapted from ref (58) in accordance with the Creative Commons Attribution License 4.0 (CC BY).

Figure 3

Single-tube RT-LAMP-integrated with CRISPR to detect SARS-CoV-2. (A) Working mechanism of RT-LAMP and CRISPR. (B) Schematic of the single-tube setup. (C) Fluorescence measurement depicting RT-LAMP amplification curves of the target N gene of SARS-CoV-2 and negative controls. (D) Cas12a-based detection of RT-LAMP amplification products. (E) Fluorescence of Cas12a-based detection at different temperatures. (F) Detection of the N gene at a range of concentrations (0–750 000 copies μL^–1) of 5 μL of sample. Adapted from ref (60) in accordance with the Creative Commons CC-BY-NC-ND license.

Figure 4

HCV detection in human plasma samples using RT-LAMP and CRISPR. (A) Schematic of the RT-LAMP and CRISPR-based platform to detect HCV. (B) LFA readout for clinical samples. (C) Fluorescence-based readout for clinical samples. (D) LFA readout of RT-LAMP-coupled CRISPR detection of HCV for 10 healthy blood donor samples, 10 HIV-infected clinical samples, and 10 HBV-infected clinical samples. (E) LOD detection of HCV genotypes 1,3, and 6 at a range of concentrations (0.01–100 ng μL^–1). Adapted from ref. in accordance with the Creative Commons CC BY license.

Figure 5

Single-tube LAMP-integrated CRISPR assay to detect K. Pneumoniae in clinically isolated sputum samples. (A) CRISPR-top workflow for DNA extraction, LAMP amplification, and CRISPR detection of K. pneumoniae in a single tube. (B) Analytical LOD of CRISPR-top and standalone LAMP. (C) Validation of CRISPR-top and standalone LAMP in clinical samples. Red indicates samples with positive results for K. pneumoniae. PC = positive control (K. pneumoniae ATCC 700603), NC = negative control, & = false-positive results, and # = false-negative results. Adapted from ref (84) in accordance with the Creative Commons Attribution 4.0 International license.