利用ATP扩增反应与生物发光法结合检测微量微生物

摘要:【目的】腺苷酸激酶（adenylate kinase, ADK）和多聚磷酸盐激酶（polyphosphate kinase, PPK）偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰，影响测定。本文旨在融合表达ADK和PPK，并建立一种方便有效的内源性ADP的去除方法，降低背景，使之与传统生物发光法结合，实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因，插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK，表达PPK-ADK融合蛋白。利用表面包裹聚胺醇（Polyurethane）的磁珠（magnetic beads），通过化学反应将腺苷酸双磷酸酶（apyrase）固定于磁珠表面，制备固相腺苷酸双磷酸酶（Beads-apyrase），用于除去与融合蛋白结合的内源性ADP，降低ATP扩增反应的背景，从而使之与生物发光反应相结合，测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性，利用Beads-apyrase可以方便而有效的去除内源性ADP，显著地降低反应背景，从而实现了利用ATP扩增反应与传统生物发光反应结合，测定了小于1 fmol的外源微量ATP，使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景，从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合，极大地提高了生物发光法的灵敏度。

Detection of low-level microorganism by concomitant use of ATP amplification and bioluminescence assay

Abstract: [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.