Disposable Static Chip Based Real-time Fluorescence Quantiative Polymerase Chain Reaction
|Keywords||Static chip PCR Real-time PCR ITO glass Disposable|
Real-time fluorescence quantitative polymerase chain reaction (Real-time FQ-PCR) is a method of quantitative analysis of DNA , its sequence - specific DNA with high sensitivity and specificity in the study of molecular biology and clinical diagnosis used widely. Desktop quantitative PCR sample and reagent dosage , more and more difficult to meet in terms of speed and flux analysis requires addition POC testing also requires the detection system portability . Therefore, quantitative PCR instrument miniaturization and quantified to become the focus of attention of the researchers . Chapter of real-time quantitative PCR research methods and PCR chip research status of the review is made on indium tin oxide (ITO) glass chip real-time fluorescence quantitative PCR for quantitative analysis of feasibility . The second chapter on the basis of existing static ITO glass thermostat chip study , the temperature control and calibration method carried out further research to improve the reliability of the PCR reaction by the method of bonding the copper foil on the chip . Chapter inverted fluorescence microscope as the basic platform initially established chip real-time PCR detection system , the excitation light source , a photomultiplier tube (PMT) and microscope operating conditions were optimized . The use of the blue light-emitting diode (LED) as the excitation light source , the light source current of 300 mA, PMT voltage 600 V , 25 times microscope objective , real-time fluorescence detection , and achieved good results . Chapter IV for the first time on the ITO glass thermostat chip based on the static temperature control mode , real-time fluorescence quantitative PCR fluorescent dye SYBR Green I the templates quantitative methods λDNA show . Through the parallel reaction and single-use method to improve throughput at the same time also effective to avoid the problem of cross-contamination . 3.8 × 105 ~ 3.8 × 108 initial copies within the linear standard curve slope of -4.9 the intercept 52.5 linear correlation coefficient of 0.9989 Ct value of the coefficient of variation of less than 5% .