[Detection of low-level microorganism by concomitant use of ATP amplification and bioluminescence assay].
OBJECTIVE
To detect low levels of microorganism by bioluminescence assay, the reaction of ATP amplification catalyzed by ADK (adenylate kinase) combined with PPK (polyphosphate kinase) can be employed. However, the endogenous ADP bound to PPK is a background source and interfere the effective detection of low levels of exogenous ATP. We expressed a fusion protein of PPK and ADK and established a new method to decrease the background signal.


METHODS
The genes of PPK and ADK were amplified by PCR and cloned into vector pET28a (+) to provide a recombinant expression plasmid pET28a (+)-PPKADK to prepare the fusion protein. Apyrase was immobilized on the surface of magnetic beads coated with polyurethane to provide Beads-apyrase to eliminate background caused by ADP bound to PPK-ADK. The exogenous ATP and microorganism were also detected by using ATP amplification reaction coupled with bioluminescence assay.


RESULTS
The purified fusion protein showed both ADK and PPK activities. Beads-apyrase could eliminate ADP contamination conveniently and effectively, thus less than 1 fmol of ATP was detected by ATP amplification reaction coupled with bioluminescence assay. Using ATP amplification reaction, the sensitivity of bioluminescence assay was 100-fold than that of normal bioluminescence assay without ATP amplification.


CONCLUSIONS
Beads-apyrase is an effective tool to eliminate the background of the reaction of ATP amplification. The sensitivity of bioluminescence assay was increased significantly with concomitant use of ATP amplification and bioluminescence assay.
Chen
Ying
Y
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. chensiyu1123@163.com
Zou
Bingjie
B
Zhu
Shuhui
S
Ma
Yinjiao
Y
Zhou
Guohua
G
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D019411:Clinical Laboratory Techniques; D008163:Luminescent Measurements / Q000379:methods*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D000263:Adenylate Kinase