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.
Highlights
In recent years the molecular amplification of polynucleotides has become increasingly important in life sciences
Since monitoring the Bioluminescent Assay in Real-Time (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
LAMP primers were designed as described in Materials and Methods complementary to sequences present on the plasmid of Chlamydia and assayed using a plasmid template synthesized to contain this sequence, referred to as Chlamydia Artificial plasmid Template (ChAT)
Summary
In recent years the molecular amplification of polynucleotides has become increasingly important in life sciences Many variants of these technologies exist, and they increasingly underpin commercial diagnostic tests as well as a large number of research applications. The full potential of these analytical tools is only realised if the analysis can detect, report and quantify the amplification occurring in a closed-tube format in real-time [1,2,3] Such assays can determine both the presence and concentration of the target in the original sample in a closed-tube format that minimises the risk of contaminating other samples with amplified DNA. The 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
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