Point-of-Care DNA Amplification for Disease Diagnosis and Management

Abstract

Early detection of pests and pathogens is of paramount importance in reducing agricultural losses. One approach to early detection is point-of-care (POC) diagnostics, which can provide early warning and therefore allow fast deployment of preventive measures to slow down the establishment of crop diseases. Among the available diagnostic technologies, nucleic acid amplification–based diagnostics provide the highest sensitivity and specificity, and those technologies that forego the requirement for thermocycling show the most potential for use at POC. In this review, I discuss the progress, advantages, and disadvantages of the established and most promising POC amplification technologies. The success and usefulness of POC amplification are ultimately dependent on the availability of POC-friendly nucleic acid extraction methods and amplification readouts, which are also briefly discussed in the review.

Changing the diagnostic paradigm for sugarcane: development of a mill-based diagnostic for ratoon stunting disease in crude cane juice

“…Previous work by our group and others have established that reducing betaine concentration in the reaction mix can significantly decrease the amplification time of the target DNA ( Zou et al., 2020 ), hence a concentration of 0.5M betaine was used in the initial Lxx LAMP assays. One of the drawbacks of isothermal LAMP amplification is its propensity to produce non-specific amplification products due to primer dimerization in the absence of DNA template ( Botella, 2022 ). Our experiments revealed that the use of 0.5M betaine occasionally produced self-amplification in no-template controls (NTCs) resulting in the production of false positives ( Supplementary Figure 3 ), therefore we performed a betaine concentration titration experiment.…”

“…Unless stated, LAMP reaction mixture contained 0.8M betaine, 20mM Tris-HCl (pH 8.8), 50mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween ® 20, 8mM MgSO 4 , 1.2mM dNTP (each), 0.32 U/mL Bst 2.0 warm start polymerase (New England Biolab), 0.2μM (each) F3 and B3 primers, 0.8μM (each) loop forward and reverse primer, 45 μL of reactions were incubated at 65°C for 60 minutes in our custom made hand held diagnostic device ( Botella, 2022 ). When necessary, the amplified products were visualized using agarose gel electrophoresis, the raw values and real time curves were analyzed using Microsoft Excel.…”

Toward Integrated Molecular Lateral Flow Diagnostic Tests Using Advanced Micro- and Nanotechnology

“…Recent advanced methods in NAATs are based on isothermal amplification (IA), which overcomes the need for thermal cycling given the ability to amplify the sequence of interest at constant temperatures. , By eliminating the need for thermocyclers, amplification techniques are set free from energy-demanding and technologically complex systems, opening the door to POCT and in-field applications. − IA techniques are thus suitable for laboratories lacking expensive PCR equipment and for low-resource settings. In addition, IA methods require less sample and reagent preparation, and the results can be read through different approaches (e.g., turbidimetry, LF), enhancing their accessibility and ease-of-use. ,, …”

Nucleic Acid Amplification‐Based Technologies (NAAT)—Toward Accessible, Autonomous, and Mobile Diagnostics

“…[ 8 ] NAATs are also important for other targets including liquid biopsies for cancers, [ 9 ] sexually transmitted infections (STIs), [ 10 ] vector‐borne diseases, [ 11 ] drug resistance in pathogens, [ 12 ] environmental DNA for surveillance of invasive species, [ 13 ] and early detection of agricultural pests and pathogens. [ 14,15 ]…”