Development of a temperature sensor array chip and a chip-based real-time PCR machine for DNA amplification efficiency-based quantification.
A temperature sensor array chip was developed to monitor the thermal cycling profiles of a polymerase chain reaction (PCR). DNA amplification efficiency of each cycle was estimated through temperature data to fit the stochastic model. A fluorescence detector system was constructed to detect the PCR amplifications of latter cycles, at which the fluorescence intensity passed the optical detection threshold. Through monitoring of both temperature and fluorescence, DNA amplification efficiency curve was completed for quantification. The Förster resonance energy transfer (FRET) was employed to detect the measurements of the PCR product amount at the reaction endpoint. The chip-based, real-time PCR machine was constructed to perform the amplification efficiency curve-based quantification method. This novel method achieved the absolute quantification of the Hepatitis B virus (HBV) DNA using a single sample without the construction of the standard curve. The coefficient of variation (CV) of the 15 replicates inter assay experiments was less than 5.87%. Compared with the CV values obtained from the commercial machine in the range of 4.33-14.56%, it is noted that CV values of the prototype with respect to the samples of different initial concentration ranging from 10(7) to 10(3)copies/ml are almost equable.
Department of Energy and Refrigerating Air Conditioning Engineering, National Taipei University of Technology, Taipei, Taiwan. f11167@ntut.edu.tw
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DS
Lee
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Chen
D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013817:Thermography / Q000379:methods*; D013821:Thermometers
D004247:DNA