应用生物发光技术定量检测PCR扩增产物

Determination of PCR product by bioluminescence

目的 建立一种基于生物发光技术的定量检测PCR产物的方法 ,并用于HBV定量PCR检测。方法 首先利用ATP硫酰化酶 (ATPsulfurylase)将焦磷酸 (PPi)定量转化成ATP ,然后利用虫荧光素酶 (luciferase)系统发光检测ATP ,从而确定样品中PPi含量。利用 2次PCR法扩增HBVDNA目的片段 ,并进行回收纯化定量 ,以之作为阳性标准模板制作标准曲线。选择适当的循环次数扩增样品中HBVDNA ,利用化学发光法检测扩增产物中PPi的量 ,进而确定样品中HBVDNA模板的量。结果 当起始模板量为 (10～ 5 )× 10 5拷贝 ,循环次数为 2 5 ,发光值与起始模板浓度呈现良好的相关。检测 32份样品血清 ,其中 2 3例HBsAg(+)、HBeAg(+)、HBcAb(+)标本中HBVDNA平均含量为 1 6× 10 5拷贝 / μl,9例HBsAg(+)、HBeAg(- )、HBcAb(+)标本中HBVDNA平均含量为 5 6× 10 3 拷贝 / μl。结论 本方法是一种操作简便。

Objective To establish a novel quantitative method for detecting PCR product based on bioluminescence.Methods\ In the assay,PPi in the samples was converted to ATP by ATP sulfurylase,then the ATP was detected by ATP bioluminescent assay kit.Target fragment of HBV DNA was amplified by Boster PCR,then purified and used as standard quantitative template to make the standard curve for sample assay.The optimum cycle number was decided to amplify HBV DNA sample.PPi in the products was detected by chemiluminescent assay,then the amount of template was determined indirectly.Results\ The relative light intensity was linearly related to template concentration in the range of (10～5)×10 5copies in optimum experimental conditions.Average virus concentrations were 5.6×10 5 copies/μl in 23 cases with HBsAg(+),HBeAg(+),HBcAb(+),1 6×10 3copies/μl in 9 cases with HBsAg(+),HBeAg(-),HBcAb(+).Conclusion This is a simple,sensitive,practical quantitative PCR method.