多重定量PCR系统中多色荧光检测和光谱串扰校正方法  

Multicolor Fluorescence Detection in the Multiplex Quantitative PCR System and Spectra Crosstalk Correction Method

作　　者：臧留琴 张镇西[1] 苗宝刚 彭年才[2] 李政[2] 

机构地区：[1]西安交通大学生命学院生物医学信息工程教育部重点实验室,陕西西安710049 [2]西安交通大学机械学院机械制造系统工程国家重点实验室,陕西西安710049

出　　处：《光学学报》2014年第1期187-193,共7页Acta Optica Sinica

基　　金：国家重大科学仪器设备开发专项(2012YQ030261);国家自然科学基金(61120106013);国家科技支撑计划(2012BAI19B057)

摘　　要：多色荧光的激发检测光路是多重定量聚合酶链式反应(PCR)系统的核心组成部分。根据系统对荧光激发的均一性、荧光检测的时间和灵敏度需求,提出一种基于磁光开关和光电倍增管(PMT)的多色荧光激发检测光路。通过四色LED单独激发和PMT检测来提高灵敏度;利用磁光效应实现光路的电控切换,配合一维扫描机构和滤光片切换装置,完成96孔标准PCR板的四色荧光扫描。该设计避免了多色荧光光路的系统串扰。由于荧光染料本身光谱特性产生的荧光光谱串扰是通过标准迭代的四维聚类分析算法计算串扰矩阵,并通过4种常用染料的实验研究,对所建荧光检测系统进行荧光光谱串扰评估。Optical design for multicolor fluorescence detection is the core part of multiplex quantitative polymerase chain reaction （PCR） system. According to the system requirements for excitation uniform, detection time and sensitivity, an optical system is presented, which is based on magneto-optical switch and photomultiplier tube （PMT） detector. Four LEDs excitation unit and PMT detection unit are adopted to improve system sensitivity. Electronic optical switch combined with the one-dimensional scanning mechanism and filter to implement four-color fluorescence scanning on a 96-well standard PCR plate. The design avoids the crosstalk among the four color optical paths. Whereas the crosstalk generated by the spectral characteristics of the fluorescent dye itself can be estimated by standard iterative four-dimensional clustering analysis algorithm. We finally evaluate the systematical fluorescent spectra crosstalk by the experimental study of four commonlv used dves.

关 键 词：光谱学 多重定量 多色荧光检测 光谱串扰校正 磁光开关 

分 类 号：O439[机械工程—光学工程]