Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

Abstract

BackgroundThe real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories.Principal FindingsHere we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required.ConclusionsThe combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings.

Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

“…These assays require only a uniform incubation temperature (i.e., are isothermal), which offers a significant reduction in complexity of instrumentation compared to that of PCR-based methods. In addition, many of the test formats can employ a wide range of detection methods, including real-time analysis (31), bioluminescence (32), fluorescent/visual dyes (33, 34), endpoint analysis via turbidimetry (35), or immunochromatographic strips (ICS) that recognize hapten-labeled amplicons (36). A further advantage of isothermal assays is that they appear to be less inhibited by confounding substances in blood, permitting the use of more-crude preparations of nucleic acids before amplification and thus simplifying sample preparation while reducing reagent requirements, cost, user training, and time to result (37–39).…”

Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification

“…Measurement of the LAMP product is typically performed by fluorescence detection of dsDNA with an intercalating or magnesium-sensitive fluorophore (4,7), bioluminescence through pyrophosphate conversion (8), turbidity detection of precipitated magnesium pyrophosphate (9,10), or even visual examination through precipitated Mg 2 P 2 O 7 or fluorescence (11,12). These methods are robust and familiar, and visual methods are ideal for use in field diagnostics, but detect total DNA amplification in a reaction and are thus limited to detection of a single target.…”

Miniaturized devices for point of care molecular detection of HIV

“…Alternatively, BART (Bioluminescent Assay in Real-Time) uses a new reporter for isothermal amplification assays. The luciferase enzyme produces transient bioluminescence during DNA synthesis [96, 97]. BART-based detection eliminates the need for an excitation source and optical filters, and avoids potential interference due to autofluorescence of reagents and chip materials.…”