【摘要】 目的基于水母发光蛋白生物发光分析技术建立一种PCR产物定量检测技术。方法利用RT-PCR对CK19-mRNA进行放大,PCR引物的5’端用生物素标记,然后用交联了地高辛寡核苷酸探针与目标生物素化的DNA模板杂交。杂交复合物与包被在微孔板上的链亲合素结合,加入标记了水母发光蛋白的地高辛抗体与地高辛特异结合,再加入钙离子激发水母发光蛋白发光,发光强度正比于CK19-mRNA的量。结果探测灵敏度可达22 amol/L的RT-PCR产物。结论该定量检测技术是一种灵敏的、可靠的、非放射性的微量CK19-mRNA定量方法。更多还原

【Abstract】 Objective A bioluminescence-based immunoassay was established for the quantitation of PCR products.Methods It amplified CK19-mRNA by using RT-PCR.The primer in the PCR reaction was labeled with a 5’ biotin molecule.A digoxigenin-conjugated oligonucleotide probe was hybridized to the target biotin-labeled DNA template.The hybridized duplex was captured onto a streptavidin-coated microtiter plate.Digoxigenin reacts with digoxigenin-specific antibodies conjugated with the photoprotein aequorin added.The amount of specific DNA captured onto the plate was quantitated by triggering the bioluminescence reaction through the addition of calcium ions.Results This technique detected as low as 22 amol/L of amplified CK19-mRNA products.Conclusion This technique is a sensitive,reliable and non-radioactive method for quantitative detection of trace CK19-mRNA.更多还原