Quantitative 'BART': Bioluminescent Assay in Real Time of nucleic acid amplification in simple handheld devices

This project aims to develop methods for quantitative determination of specific nucleic acids, and hence any specific organism, in a portable device. The project will use a new detection method based on using a thermostable luciferase to emit light as a specific DNA sequence is amplified. BART (bioluminescent assay in real-time) follows the accumulation of pyrophosphate (in a sample) that necessarily occurs as dNTPs are incorporated into the amplified DNA. As such, the level of pyrophosphate indicates the extent of amplification and hence the presence or not (and quantity) of target nucleic acid in the sample. The biochemistry of BART is an adaptation of the 'Enzymatic Luminometric Inorganic pyrophosphate Detection Assay, or 'ELIDA', in which the pyrophosphate is converted into ATP by ATP sulphurylase in the reaction with APS (adenosine 5'-phsophosulphate): PPi + APS -> ATP The ATP produced is then detected by firefly luciferase in the presence of its substrate luciferin: ATP + luciferin -> AMP + PPi + oxyluciferin + light and the light is readily monitored by CCD camera or light detecting diode. This reaction is known as BART, and was invented by Dr L. Tisi and Prof. Jim Murray, the two proposed supervisors of this project. BART is protected by granted EU and US patents and Lumora has sole rights to this technology. It depends on the use of a thermostable luciferase, also invented by Tisi and Murray. BART is used in conjunection with isothermal amplification, which occurs not as a result of temperature cycling but through the use of strand displacing polymerases such as Bst DNA polymerase that extend from primers that provide stringent amplification specificity in a reaction that occurs at a constant 56-65 C, obviating the energy requirements needed for rapid temperature cycling. Loop-Mediated Amplification (LAMP) is one such example (Notomi et al., 2000 Nucleic Acids Res. 2000 Jun 15;28:E63). LAMP is characterised by the use of primers capable of forming loops and isothermally amplifies DNA targets with high specificity even without confirmatory probes. This is a reflection of the amplification mechanism which requires four independent target sites to be recognized for amplification to occur, compared to just two in PCR. LAMP has become a very well characterized method with high specificity and sensitivity and has been validated for diagnostic products. LAMP is one of a number of isothermal amplification technologies that can be interfaced with BART. Lumora has successfully established a single tube system for coupled DNA amplification with BART, and developed both bench top and portable devices for monitoring the reactions. It has been successfully demonstrated for detection of Chalmydia, GMOs, and a range of food microorganisms and has been through validation trials. Extremely simple preparation methods have been developed, and the combined assay shows greater robustness than RT-PCR to contaminants from the sample. This project aims to tackle a remaining challenge of BART based detection systems, which is to achieve a high level of quantitative accuracy in portable devices. This is required for a number of applications, including the determination of GM levels in agricultural products and foodstuffs to comply with EU requirements and viral load determination in clinical applications. BART outputs are proportional to input DNA, but a detailed analysis of the parameters affecting quantitation, the development of appropriate controls, the development of optimal purification systems for DNA in the field/ clinic and its combined use in a hand held device are the challenges to be met in this project. Quantitation will be achieved by using both known standards and statistical probability methods based on yes-no detection of samples subject to dilution, as developed for PCR by Arne Holst-Jensen, a collaborator in the EU Co-Extra network in which Lumora is also a partner.