Chemical Trends in Sample Preparation for Nucleic Acid Amplification Testing (NAAT): A Review

Abstract

Nucleic acid amplification testing facilitates the detection of disease through specific genomic sequences and is attractive for point-of-need testing (PONT); in particular, the early detection of microorganisms can alert early response systems to protect the public and ecosystems from widespread outbreaks of biological threats, including infectious diseases. Prior to nucleic acid amplification and detection, extensive sample preparation techniques are required to free nucleic acids and extract them from the sample matrix. Sample preparation is critical to maximize the sensitivity and reliability of testing. As the enzymatic amplification reactions can be sensitive to inhibitors from the sample, as well as from chemicals used for lysis and extraction, avoiding inhibition is a significant challenge, particularly when minimising liquid handling steps is also desirable for the translation of the assay to a portable format for PONT. The reagents used in sample preparation for nucleic acid testing, covering lysis and NA extraction (binding, washing, and elution), are reviewed with a focus on their suitability for use in PONT.

Method for lysis and paper-based elution-free DNA extraction with colourimetric isothermal amplification

“…However, amplicons were detected by gel electrophoresis following LAMP but not PCR, indicating the paper inhibited both amplification and detection during qPCR but only interfered with the fluorescence detection in qLAMP. Combined with an expected loss in sensitivity at higher salt levels because of an increase in dissociation constant (K d ) of SYBR green for DNA 1 , 29 , fluorescence detection was deemed unsuitable for the targeted extraction following PASAP. cLAMP has become a popular choice for in-field testing for non-quantitative detection 31 .…”

“…The results presented above suggest that the eluting strength of 70% ethanol may be too high for it to be a suitable washing reagent. Washing reagents during DNA extraction on anionic stationary phases have included water-miscible solvents like isopropanol to remove the salt 1 . Non-polar solvents were used in the two-phase wash to limit the carryover of aqueous inhibitors but were typically added between the wash step and the final elution 28 .…”

“…Nucleic acid amplification tests (NAATs) have gained popularity due to their high sensitivity and specificity for identifying target species in samples of interest. Research has focused on integrating NAAT into portable devices to meet the increasing demand for point-of-need testing (PONT) 1 . In parallel, paper analytical devices (PADs) have been demonstrated to provide an affordable substrate for chemical and biological assays due to accessible manufacture, low cost, and ease of disposal [2][3][4][5] .…”