LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

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Abstract

Seeking optimized infectious pathogen detection tools is of primary importance to lessen the spread of infections, allowing prompt medical attention for the infected. Among nucleic-acid-based sensing techniques, loop-mediated isothermal amplification is a promising method, as it provides rapid, sensitive, and specific detection of microbial and viral pathogens and has enormous potential to transform current point-of-care molecular diagnostics. In this review, the advances in LAMP-based point-of-care diagnostics assays developed during the past few years for rapid and sensitive detection of infectious pathogens are outlined. The numerous detection methods of LAMP-based biosensors are discussed in an end-point and real-time manner with ideal examples. We also summarize the trends in LAMP-on-a-chip modalities, such as classical microfluidic, paper-based, and digital LAMP, with their merits and limitations. Finally, we provide our opinion on the future improvement of on-chip LAMP methods. This review serves as an overview of recent breakthroughs in the LAMP approach and their potential for use in the diagnosis of existing and emerging diseases.

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Loop-mediated isothermal amplification of DNA.

T. Notomi (2000)

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

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CRISPR-Cas12–based detection of SARS-CoV-2

James Broughton, Xianding Deng, Guixia Yu … (2020)

An outbreak of betacoronavirus SARS-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from US patients, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US CDC SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.

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Nucleic acid detection with CRISPR-Cas13a/C2c2

Jonathan S. Gootenberg, Omar O Abudayyeh, Jeong Wook Lee … (2017)

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed SHERLOCK ( S pecific H igh Sensitivity E nzymatic R eporter Un LOCK ing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.