The LiMA Technology: Measurement of ATP on a Nucleic Acid Testing Platform

Abstract

Detection of bacterial growth is used in the diagnosis and treatment of infectious disease, blood screening, food safety, product quality assurance, and life science research. The measurement of intracellular ATP content has long been the standard for rapid bacterial growth and viability measurement (1)(2)(3). The current methods used to detect ATP include luminescence generated by the enzyme system firefly luciferase/luciferin. The chemistry involved in this process is simple and can be used with a wide range of luminescence equipment, from handheld devices to sophisticated, laboratory-based instruments for high-throughput applications(4). We have developed an alternative, quantitative assay method for ATP based on a nucleic acid testing (NAT) format. During the past decade NAT has become the method of choice for bacterial identification (5). NAT hardware, such as thermal cyclers, isothermal instruments, and real-time/kinetic product detection systems, are now commonplace laboratory equipment. The new method, LiMA (Ligase Mediated ATP Amplification Assay)(6), uses DNA ligase, an ATP-requiring enzyme(7), to join 2 oligonucleotides in a nicked-DNA substrate and create a template that can be amplified in a DNA amplification reaction (Fig. 1⇓ ). Before the LiMA process, the ligase is treated with pyrophosphate to ensure that all the enzyme molecules are in the deadenylated form. Thus the ligase is inactive until it binds a molecule of ATP, which leads to the loss of the pyrophosphate moiety from ATP and the formation of a covalent enzyme—AMP intermediate linked to a lysine side-chain in the enzyme. In this reaction the enzyme becomes charged by an ATP molecule; essentially, a sample is scavenged for its ATP content. In the next step of the reaction the AMP nucleotide is transferred to the 5′ phosphate of the nicked-DNA strand, followed by attack on the AMP-DNA bond by the 3′-OH of …