Research On Fluorescence Detection Technologies Of Ouantitative PCR

Quantitative polymerase chain reaction (Q-PCR) is a technology based on fluorescence resonance energy transfer (FRET) that accounts for the addition of fluorophore into PCR reagent so that the change of amplification product copy number in each thermal cycle can be monitored through real time accumulation of fluorescence emission. For accurate quantification of amplification copy number, not only the reaction time and temperature of PCR reagents should be rigidly controlled, but also the quantitative analysis of fluorescent signals emitted during the reaction should be conducted accurately including an optical system that can detect signals as weak as microampere and a fluorescence signal monitoring system that is used to quantitatively describe collected fluorescence data.A Q-PCR instrument always consists of a thermal cycler system which can amplify target sequences rapidly and a fluorescence detection system that can quantify target sequences accurately. Amongst, the optical system used for fluorescence detection is the key component. Based on conventional confocal fluorescence detection optical structure, a γ-type optical fiber system is proposed in this paper. Through total internal reflection (TIR), this optical fiber system receives the excitation light at the incident end of the γ-type fiber and excites the fluorescence reagent in reaction tubes to emit fluorescent signals which will be transmitted to a photoelectric sensor through the other branch of the fiber optic. Due to fewer introductions of traditional optical devices, the power depletion can be largely reduced during light transmission process.Factors like light source, lens parameters, NA for optical fiber and fiber loss that affect the light transmission efficiency of γ-type fiber optical system are analyzed by ZEMAX. Combined with fluorescence detection experiment, the validity of γ-type fiber optical system has been verified through comparison of confocal and γ-type fiber based fluorescence detection system experiment results.The optical system detects the 96-hole sample block through scanning. To avoid structural interference between the optical system and the thermal cycler system, two orthogonal ball screw electric tables are adopted to drive the optical module and thermal cycler module respectively so that the fluorescence can be excited and emitted.Fluorescence captured by the optical system is very weak, normally in the magnitude of several microamperes and mixed with noise, so it is necessary to amplify and filter the collected fluorescence data before conversion from analog signal to digital signal and transmission through data acquisition card to the upper data processing system afterwards. To implement the accurate quantification of target sequences, the data processing system needs to eliminate the residual noise with a digital filter and improve the curve fitting result by the smoothing method of five points. Finally, quantitative detection of target sequences can be completed since the amplification copy number of each thermal cycle and starting copy number can be calculated according to the loaded quantification algorithm in advance.Based on the sigmoidal curve-fitting method presented by Liu & Saint, an improved quantification algorithm was proposed. This improved algorithm takes into consideration the dynamics of thermal cycle efficiency during the polymerase chain reaction so that it would be more objective to fit the collected fluorescence data.It can be obtained from fluorescence detection and gene quantification experiments conducted with the Q-PCR instrument developed by our research group that the optical system based on γ-type optical fiber can improve the fluorescence detection sensitivity effectively and fitting the fluorescence data with the improved quantification algorithm increase the reproducibility and reliability of quantification results.