Development of high throughput Dual Laser system incorporating CCD-based multichannel Czerny – Turner spectrometer for real-time qPCR fluorescence intensity measurements

Abstract

In this article, the development of a commercial high throughput system incorporating a multichannel CCD-based Czerny – Turner spectrometer for real-time fluorescence quantitative polymerase chain reaction (qPCR) measurements is described. The system employs two 20 mW lasers (532 ± 1 nm and 637 ± 3 nm) 2.5× Galilean beam expander-focuser, 5×4-200 μm core diameter fiber-optic bundle and a two-axis galvanometer for exciting fluorescent dyes. Further, the optical measurement system can detect twenty reaction vessels in asynchronous manner and up to five different fluorescent dyes (5-plex) at 1 nM dye concentration in each of the reaction vessels simultaneously. Moreover, PCR curves obtained using the optical measurement system for a genomic deoxyribonucleic acid (DNA) template containing three fluorescent generic binding dyes (FAM, Q670 and CRF610) is discussed. The measurement system repeatability is <1% CV (coefficient of variance). Finally, a performance comparison between the reported qPCR laser-based measurement system and an LED-based qPCR measurement system described by the author in ref. 14 is provided.