The fluorescence detection system of the real-time fluorescence quantitative PCR (qPCR) instrument is the core component for nucleic acid quantification. Its working principle revolves around real-time monitoring of changes in fluorescence signals during PCR amplification. By correlating fluorescence intensity with the amount of amplified product, quantitative analysis of the initial template is achieved. The following is a detailed analysis of its working mechanism:

1、 The core components of a fluorescence detection system

The fluorescence detection system mainly consists of three parts: excitation light source, optical filter group, and detector. The three work together to complete the "excitation filtering capture" process of fluorescence signals:

Excitation light source: providing light of a specific wavelength (such as xenon lamp, LED lamp, or laser) for exciting fluorescent substances in the reaction system.

Optical filter group: including excitation filter and emission filter, which respectively filter out excitation light and fluorescence of specific wavelengths, reduce background interference, and ensure signal specificity.

Detector: usually a photomultiplier tube (PMT) or charge coupled device (CCD), which converts the captured fluorescence signal into an electrical signal and then converts it into a digital signal through software.

2、 The generation of fluorescent signals: dependent on fluorescent labeling substances

The premise of fluorescence detection is the presence of excitable fluorescent substances in the PCR reaction system, whose fluorescence intensity varies with the increase of PCR product quantity. Common types include:

SYBR Green I and other DNA binding dyes

Characteristics: It can be non specifically embedded into minor grooves of double stranded DNA (dsDNA). When unbound, the fluorescence signal is extremely weak, but after binding, the fluorescence intensity is significantly increased (about 1000 times).

Signal changes: With PCR amplification, the dsDNA product increases, the dye binding amount increases, and the fluorescence signal synchronously strengthens.

Hydrolysis probe (TaqMan probe)

Structure: An oligonucleotide probe complementary to the target sequence, labeled with a 5 'end fluorescent reporter group (such as FAM, VIC) and a 3' end fluorescent quenching group (such as TAMRA).

Principle: When the probe is intact, the distance between the reporting group and the quenching group is close, and the fluorescence is quenched (FRET effect); During the PCR extension stage, the 5 '→ 3' exonuclease activity of Taq enzyme hydrolyzes the probe, releases the reporter group, and enhances the fluorescence signal (positively correlated with the product quantity).

Hybrid probes (such as Molecular Beacons, Scorpions probes)

Principle: The probe folds itself to form a stem loop structure, bringing the reporting group closer to the quenching group (without fluorescence); When the probe hybridizes with the target sequence, the stem loop structure opens, the functional groups separate, and the fluorescence signal is released.

3、 Detection process: Real time synchronous monitoring of amplification process

The fluorescence detection system runs synchronously with the PCR thermal cycle process, and completes signal acquisition after the extension stage of each amplification cycle (or at a specific temperature). The specific steps are as follows:

Excitation light irradiation

When the PCR cycle enters the signal acquisition stage (usually after annealing or extension), the excitation light source emits light of a specific wavelength (such as 490 nm for exciting FAM fluorescence), which is filtered through an excitation filter and accurately irradiated onto the sample in the reaction well.

Fluorescent substances emit light

Fluorescent substances in the reaction system (such as SYBR Green I bound to dsDNA or hydrolyzed TaqMan probes) absorb excitation light energy, transition from the ground state to the excited state, and release specific wavelength fluorescence when returning to the ground state (such as FAM emitting 520 nm fluorescence).

Fluorescence signal filtering and capture

The emission filter only allows target fluorescence (such as 520 nm) to pass through, filtering out background signals such as unabsorbed excitation light and sample spontaneous fluorescence to ensure signal purity.

The detector (such as PMT) captures the filtered fluorescence signal, converts it into an electrical signal (current or voltage), amplifies it through the instrument's built-in circuit, and transmits it to the data processing system.

Signal recording and analysis

The software converts electrical signals into fluorescence intensity values (RFU, relative fluorescence units) and displays them in real-time in the form of a "cycle number fluorescence intensity" curve. As PCR amplification proceeds, the fluorescence intensity gradually increases. When the set threshold is reached, the corresponding cycle number (Ct value) is recorded for subsequent quantitative analysis.

4、 Key technology: Ensure detection sensitivity and accuracy

Multi channel detection capability

Modern qPCR machines typically support multi-channel detection (such as 4-6 channels), and by equipping filter groups with different wavelengths, multiple fluorescent markers (such as FAM, VIC, ROX) can be detected simultaneously, achieving multiplex PCR reactions (amplifying multiple target genes in the same reaction tube).

Uniformity temperature control and signal acquisition

Fluorescence detection needs to be accurately matched with the temperature of PCR thermal cycling (such as collecting signals at 60 ℃ annealing temperature), so the thermal cycling module of the instrument needs to have the ability to quickly raise and lower temperatures and a uniform temperature distribution (within ± 0.2 ℃) to avoid signal deviation caused by temperature differences.

Background signal suppression

By optimizing filter bandwidth (such as narrowband filters), using low fluorescence background reaction consumables (such as optical grade 96 well plates), and designing a darkroom inside the instrument, non-specific fluorescence interference is minimized to the greatest extent possible, and detection sensitivity is improved (as low as single copy genes).

5、 The correlation with quantitative analysis: the significance of Ct values

The core goal of a fluorescence detection system is to obtain the Ct value (Cycle threshold), which is the number of amplification cycles when the fluorescence intensity reaches the set threshold. The more initial templates there are, the fewer cycles the fluorescence signal reaches the threshold (the smaller the Ct value), and vice versa, the larger the Ct value. By comparing the Ct value of the unknown sample with the Ct value of the standard, the concentration of the initial template can be calculated.

summarize

The fluorescence detection system of the real-time fluorescence quantitative PCR instrument tracks the fluorescence changes during the PCR amplification process in real time through the process of "excitation light → fluorescence substance emission → signal filtering → detection conversion". Its core is to convert the "quantitative change" of nucleic acid amplification into the "qualitative change" of fluorescence signal, and achieve accurate quantification through Ct value. The sensitivity, specificity, and stability of the system directly determine the reliability of qPCR results and are key technical supports in fields such as molecular biology and clinical diagnosis.