How do Real-Time PCR work?
A real-time PCR system pairs a Peltier or air-heated thermal block with an excitation source, filters and a detector. Reactions contain a fluorescent reporter: either an intercalating dye such as SYBR Green that binds double-stranded DNA, or sequence-specific probes such as hydrolysis (TaqMan-type) or molecular beacon probes. Excitation light from LEDs or halogen lamps passes through a filter set, and emission is read by a photodiode, PMT or CCD at the end of each annealing or extension step. The software plots fluorescence against cycle number and assigns a quantification cycle, Cq or Ct, where the signal crosses a threshold. Cq relates inversely to the logarithm of starting copy number, enabling standard-curve or relative quantification. Melt curve analysis adds product specificity checks.
Which Real-Time PCR variants are available?
- 96-Well Real-Time PCR System — A 96-well real-time PCR system is the standard qPCR platform, reading four to six fluorescence channels from a 96-well plate with Peltier-controlled temperature.
- 384-Well Real-Time PCR System — A 384-well real-time PCR system reads a full 384-well plate with whole-plate imaging or fast scanning, with reaction volumes of 5–20 µL.
- Multiplex 6-Channel Real-Time PCR System — A six-channel multiplex real-time PCR system separates up to six fluorophores in each well using narrow excitation and emission filter sets.
- Rotor-Type Real-Time PCR Cycler — A rotor-type real-time PCR cycler spins 36 to 100 tubes in a circular rotor heated and cooled by air, reading each tube as it passes the optical head.
- Portable Real-Time PCR Analyzer — A portable real-time PCR analyzer is a battery-capable, suitcase-sized qPCR unit with two to four channels and 8 to 48 wells, built for on-site pathogen and contamination screening.
How do I choose the right variant?
Decide first on throughput and format: 96-well suits most laboratories, 384-well suits high-volume screening, and rotor or portable designs suit small batches. Then match the number of optical channels to your multiplex needs, since four channels cover most dual- or triplex assays with an internal control, while five or six channels support larger panels. Check that the excitation and emission filters fit your dyes, including FAM, HEX/VIC, ROX and Cy5. Compare thermal uniformity and ramp rate, detection sensitivity at low copy number, and software features such as absolute quantification, melt curves and gene-expression analysis. For regulated diagnostics, confirm audit trail, user levels and data-export options, and ask whether IVD registration support exists.
Where are Real-Time PCR used?
Real-Time PCR are specified in clinical pathogen nucleic acid testing, gene expression analysis by RT-qPCR, food and GMO testing, environmental and water microbiology and copy-number and SNP analysis.
Which standards and certifications apply?
Work with Real-Time PCR is commonly governed by ISO 20395, ISO 22174, CLSI MM06, CLSI MM19, IEC 61010-1, IEC 61010-2-101 and MIQE guidelines (research reporting). Instrument certifications such as CE marking are confirmed per configuration and never assumed.
What are the key considerations before purchase?
Install the instrument on a level, vibration-free bench away from sunlight, with ambient temperature of 15–30 °C and stable mains. Perform optical calibration with the supplier's dye or ROX calibration plates at installation and after lamp or LED replacement. Verify thermal uniformity annually. Use optically clear, low-fluorescence plates and seals that match the well format, and avoid fingerprints on the seal. Keep pre- and post-PCR areas separate and never open amplified plates near the instrument. Clinical laboratories should plan for software validation, access control and audit trail in line with their quality system, and keep backups of run files and melt-curve data.
