Abstract
Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loop-mediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result.
Highlights
Disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens
It is important to note that the concentrations of loop-mediated isothermal amplification (LAMP) reagents such as magnesium sulfate, deoxy nucleoside triphosphates (dNTPs), and betaine may be optimized for maximum assay sensitivity and efficiency (Njiru et al, 2011)
Unlike some intercalating dyes that must be added after amplification is complete, such as SYBR Green, hydroxynaphthol blue (HNB) can be present in the initial LAMP reaction solution
Summary
HDA is an isothermal amplification method introduced in 2004 by the Kong group (Vincent et al, 2004). HDA uses helicase, an enzyme that unwinds DNA duplexes during DNA replication, removing the need for denaturation via thermocycling (Cao et al, 2013). Thermophilic HDA (tHDA) is the most common type of HDA used by researchers, which uses thermostable Tte-UvrD helicase with Bst-DNA polymerase and operates at a single uniform temperature between 60 and 65 ◦C (An et al, 2005). Mesophilic HDA (mHDA) is another type of HDA that uses E. coli UvrD helicase, operates at 37 ◦C, and yields lower specificity than tHDA (An et al, 2005; Vincent et al, 2004). The HDA primer sequences need to be carefully designed, and many primer combinations must be screened, which adds additional time and cost (Yang et al, 2015; Deng and Gao, 2015)
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