Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

Isothermal Amplification of Nucleic Acids

Introduction A Why POC Diagnostics? B Time B Patient Responsibility and Compliance B Cost B Diagnostic Targets C Proteins C Metabolites and Other Small Molecules C Nucleic Acids C Human Cells D Microbes/Pathogens D Drugs and Food Safety D Current Context of POC Assays E POC Glucose Assays E Lateral Flow Assays E Limitations of “Traditional” POC Approaches F Enabling Technologies G Printing and Laminating G Microfluidic Technologies and Approaches: “Unit Operations” for POC Devices G Pumping and Valving H Mixing I Separation I Reagent Storage J Sample Preparation K Surface Chemistry and Device Substrates L Physical Adsorption L Bioaffinity Attachment L Covalent Attachment M Substrate Materials M Detection M Electrochemical Detection N Optical Detection N Magnetic Detection N Label-Free Methods O Enabling Multiplexed Assays O Recent Innovation O Lateral Flow Assay Technologies O Proteins P Antibodies P Protein Expression and Purification Q Nucleic Acids Q Aptamers R Infectious Diseases and Food/Water Safety R Blood Chemistry S Coagulation Markers S Whole Cells S Trends, Unmet Needs, Perspectives T Glucose T Global Health and the Developing World T Personalized Medicine and Home Testing U Technology Trends U Multiplexing V Author Information V Biographies V Acknowledgment W References W

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Point of care diagnostics: Status and future

Before the emergence of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2003, only 12 other animal or human coronaviruses were known. The discovery of this virus was soon followed by the discovery of the civet and bat SARS-CoV and the human coronaviruses NL63 and HKU1. Surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. The explosive nature of the first SARS epidemic, the high mortality, its transient reemergence a year later, and economic disruptions led to a rush on research of the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the virus and the disease. This research resulted in over 4,000 publications, only some of the most representative works of which could be reviewed in this article. The marked increase in the understanding of the virus and the disease within such a short time has allowed the development of diagnostic tests, animal models, antivirals, vaccines, and epidemiological and infection control measures, which could prove to be useful in randomized control trials if SARS should return. The findings that horseshoe bats are the natural reservoir for SARS-CoV-like virus and that civets are the amplification host highlight the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.

Severe Acute Respiratory Syndrome Coronavirus as an Agent of Emerging and Reemerging Infection

https://link.springer.com/content/pdf/10.1007%2Fs10156-009-0669-9.pdf

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

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SHERLOCK: nucleic acid detection with CRISPR nucleases.

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30-60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.

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Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects

https://link.springer.com/content/pdf/10.1007%2Fs10156-013-0590-0.pdf

Loop-mediated isothermal amplification (LAMP): recent progress in research and development

(See the major article by Hopkins et al on pages 645–57.)

Since the 1880s, the standard diagnostic test for malaria has been microscopic examination of peripheral blood smears [1–3]. The development of rapid antigen-detection tests (RDTs) in the early 1990s has improved diagnosis of Plasmodium infection in malaria-endemic countries and targeting of treatment [4, 5]. In countries where malaria is not endemic, where imported malaria cases in travelers can be mistaken for nonspecific viral illness [6, 7], causing delay in diagnosis and in some cases progression to severe illness and death [8–11], RDTs have improved the capacity for rapid malaria diagnosis by nonspecialist health workers [12–15]. While RDTs and expert microscopy are considered adequate for case management in malaria-endemic populations [16], there is growing interest in “improvements in point-of-care tests for case management, and the development of new tests capable of identifying very low parasite densities in asymptomatic individuals in field settings for mass screening and treatment” [17].

Polymerase chain reaction (PCR), in its most sensitive form, has a limit of detection as low as 50 parasites per milliliter of peripheral blood [18, 19], although results take 10–16 hours, whereas expert microscopy is less sensitive but routinely produces results in 60 minutes. Real-time quantitative PCR (qPCR) can reliably detect parasite DNA within 3–5 hours of sample receipt but is also less sensitive than nested PCR [20]. Recent studies show that loop-mediated amplification (LAMP) assays for malaria, which deploy isothermal molecular amplification in a closed system with visual readout, can deliver PCR-level diagnostic accuracy in a little more than 1 hour, with lower laboratory capacity requirements [21–23]. To date, malaria LAMP has not been available in a format suited to routine diagnosis in the clinic. A clinically validated, CE-marked assay would be an attractive alternative to RDT and microscopy in settings where malaria is or is not endemic.

A malaria LAMP kit has been developed as a point-of-care diagnostic test, and after CE marking it was released commercially in mid-2012. This kit comprises a disposable extraction device and tubes containing vacuum-dried and temperature-stable reaction components. We investigated the diagnostic accuracy of this kit for case management in a study of sequential blood samples from suspected imported malaria cases received by a specialist parasitology laboratory in London, United Kingdom, over the first 7 months of 2011. Primary diagnosis was by expert microscopic examination of blood films. An established nested PCR assay was deployed as the reference standard [18]. Diagnostic accuracy of the new kit was superior to that of microscopy and similar to that of nested PCR but with the additional benefits of reduced assay time and ease of operation.

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Hidetoshi Kanda

Papers

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects

Loop-mediated isothermal amplification (LAMP): recent progress in research and development

Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria

Validation of the Loop-Mediated Isothermal Amplification Method for Single Nucleotide Polymorphism Genotyping with Whole Blood