ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell.
We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.
Satoh
Tetsuya
T
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University.
Kato
Junichi
J
Takiguchi
Noboru
N
Ohtake
Hisao
H
Kuroda
Akio
A
D000255:Adenosine Triphosphate / Q000032:analysis*; D000263:Adenylate Kinase; D001419:Bacteria / Q000166:cytology / Q000302:isolation & purification*; D004926:Escherichia coli / Q000166:cytology / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D008156:Luciferases; D008163:Luminescent Measurements / Q000379:methods* / Q000592:standards; D017853:Phosphotransferases (Alcohol Group Acceptor); D011993:Recombinant Fusion Proteins
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C475691:polyphosphate kinase, E coli; D000263:Adenylate Kinase