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

Date Published: November 30, 2010

Publisher: Public Library of Science

Author(s): Olga A. Gandelman, Vicki L. Church, Cathy A. Moore, Guy Kiddle, Christopher A. Carne, Surendra Parmar, Hamid Jalal, Laurence C. Tisi, James A. H. Murray, Paulo Lee Ho.

Abstract: 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.

Here 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.

The 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.

Partial Text: 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. Most diagnostic applications rely on detection of a target nucleic acid through the process of amplification whose specificity is determined by the use of oligonucleotide primers complementary to the target sequence. 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]–[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.

Molecular diagnostic tests provide the “gold standard” in terms of sensitivity and specificity, and are in principle capable of detecting single copies of a specific nucleic acid sequence in a sample through the process of repeated copying by nucleic acid amplification. There is a rapidly increasing demand for such molecular diagnostic tests driven by the requirement for sensitive and accurate determination of contaminating or disease organisms, the presence of adventitious genetic material, or the diagnosis of genetically determined disease states. In particular, there is a need for tests providing speed, simplicity and robustness in both molecular assay and the necessary equipment. Such attributes are also vital in the low resource settings of the developing world, where molecular diagnostics have yet to have a widespread impact.