Introduction
Quantitative real-time PCR (qPCR) is one of the most core quantitative detection technologies in the molecular diagnostics field. Building upon traditional PCR, it adds a fluorescence monitoring system that enables real-time detection of product accumulation during amplification, allowing accurate quantification of target nucleic acids. From infectious disease viral load monitoring (HIV, HBV, CMV) to gene expression analysis (mRNA, miRNA), from copy number variation detection to pathogen quantification, qPCR technology has become an indispensable tool in clinical diagnostics and life science research due to its high sensitivity, wide dynamic range, and operational convenience. The performance of high-quality qPCR reagents heavily depends on the proper selection and combination of hot-start DNA polymerases, dNTPs, fluorescent probes/dyes, passive reference dyes (ROX), buffers, stabilizers, and other raw materials. As the third article in the molecular diagnostics raw material series (the second after the general outline), this article systematically introduces the technical principles, main application areas, fluorescence chemistry systems, key raw material selection points, formulation examples, and frequently asked questions for qPCR, providing a complete raw material technical reference for qPCR reagent development.
I. Overview of qPCR Technology
1.1 Basic Principle of qPCR
qPCR adds a fluorescence monitoring system to the thermal cycling of traditional PCR. At the end of each cycle's extension phase (or at the end of annealing/extension), the instrument collects fluorescence signals, recording the change in fluorescence intensity with cycle number, generating an amplification curve. By analyzing the Ct value (cycle threshold — the cycle number at which fluorescence signal reaches a set threshold), quantification of the starting template is achieved.
qPCR Quantification Principle:
| Parameter | Explanation | Relationship |
|---|---|---|
| Ct Value | Cycle number at which fluorescence reaches threshold | Ct ∝ -log(starting template amount) |
| Starting Template Amount | Quantity of target nucleic acid at reaction start | Higher amount = lower Ct value |
| Standard Curve | Ct values vs known concentration standards | Used for absolute quantification |
1.2 qPCR vs Traditional PCR Comparison
| Feature | Traditional PCR | qPCR |
|---|---|---|
| Detection Method | End-point detection (gel electrophoresis) | Real-time detection (fluorescence monitoring) |
| Quantification Capability | Semi-quantitative/qualitative | Absolute/relative quantification |
| Dynamic Range | 10²-10³ | 10⁶-10⁹ |
| Sensitivity | Medium | High (1-10 copies/reaction) |
| Detection Time | 1.5-3 hours | 1-2 hours |
| Post-Processing | Required (gel electrophoresis) | Not required |
| Contamination Risk | Higher (tube opening) | Lower (closed-tube detection) |
| Throughput | Low-medium | High (96/384-well plates) |
1.3 Main Application Areas of qPCR
| Application Area | Specific Test Items | Format | Clinical/Application Significance |
|---|---|---|---|
| Viral Load Monitoring | HIV, HBV, HCV, CMV, EBV | Absolute quantification | Antiviral therapy efficacy monitoring |
| Gene Expression Analysis | mRNA, miRNA, lncRNA quantification | Relative quantification (ΔΔCt) | Biomarker discovery, mechanism research |
| Pathogen Detection & Quantification | SARS-CoV-2, Influenza, RSV | Absolute quantification | Infectious disease diagnosis |
| Copy Number Variation (CNV) Analysis | Gene copy number changes | Relative quantification | Genetic disease diagnosis |
| Tumor Marker Detection | Fusion genes, mutation quantification | Absolute/relative quantification | Companion diagnostics |
| GMO Detection | Transgene copy number | Absolute quantification | Food safety |
1.4 qPCR Quantification Methods
| Quantification Method | Principle | Advantages/Disadvantages | Application Scenarios |
|---|---|---|---|
| Absolute Quantification | Standard curve of Ct vs concentration; interpolate unknown samples | Accurate, requires standards | Viral load, pathogen quantification |
| Relative Quantification (ΔΔCt Method) | Compare target gene Ct to reference gene; calculate relative expression change | No standards needed, simple operation | Gene expression analysis |
| Relative Quantification (Standard Curve Method) | Standard curve for target and reference | More accurate than ΔΔCt | Gene expression analysis |
| Digital PCR (dPCR) Assisted | End-point dilution, absolute quantification, no standard curve needed | Extremely high precision, higher cost | Rare mutation detection |
II. qPCR Fluorescence Chemistry Systems
qPCR fluorescence signals originate from two main chemistry systems: DNA-binding dyes and fluorescent probes.
2.1 DNA-Binding Dyes (SYBR Green Method)
Principle: SYBR Green I is an asymmetric cyanine dye. Free dye has weak fluorescence, but upon binding to double-stranded DNA (dsDNA), fluorescence is enhanced more than 1000-fold. As PCR product accumulates, dsDNA increases, and fluorescence signal increases.
| Feature | SYBR Green I | EvaGreen | SYTO Series |
|---|---|---|---|
| Excitation/Emission (nm) | 497/520 | 500/530 | Dye-dependent |
| Fluorescence Enhancement | >1000× | >1000× | 200-500× |
| PCR Inhibition | Low | Very low | Very low |
| Thermal Stability | Moderate (protect from light) | Good | Good |
| Application Scenarios | Routine qPCR | High-resolution melting curve | Multiplex qPCR |
SYBR Green Method Advantages:
-
Low cost (no probe synthesis required)
-
Can perform melting curve analysis (differentiates non-specific products)
-
Suitable for primer screening and system optimization
SYBR Green Method Disadvantages:
-
Lower specificity (non-specific products also generate signal)
-
Cannot perform multiplex detection (single channel)
-
Sensitive to primer dimers
2.2 Fluorescent Probes (TaqMan Method)
Principle: A TaqMan probe is an oligonucleotide labeled with a fluorescent reporter at the 5' end (e.g., FAM) and a quencher at the 3' end (e.g., BHQ1). In the intact probe, reporter fluorescence is absorbed by the quencher. During PCR extension, the 5'→3' exonuclease activity of Taq polymerase cleaves the probe, separating the reporter from the quencher, generating fluorescence signal. Fluorescence intensity is proportional to the amount of amplified product.
Common Fluorescent Reporter and Quencher Combinations:
| Fluorescent Reporter | Excitation/Emission (nm) | Recommended Quencher | Typical Use |
|---|---|---|---|
| FAM | 494/518 | BHQ1, TAMRA | Single-plex or lowest wavelength channel in multiplex |
| VIC / HEX | 535/554 | BHQ1 | Second channel in multiplex detection |
| ROX | 575/602 | BHQ2 | Multiplex detection, passive reference dye |
| CY5 | 650/670 | BHQ2, BHQ3 | Far-red channel in multiplex |
| TAMRA | 565/580 | BHQ2 | Traditional probe (self-quenching) |
TaqMan Method Advantages:
-
High specificity (probe hybridization adds an extra layer of specificity)
-
Can perform multiplex detection (different fluorescence channels)
-
High data quality (low background, high signal-to-noise ratio)
TaqMan Method Disadvantages:
-
Higher cost (probe synthesis required)
-
Cannot perform melting curve analysis
-
Probe design requires optimization (Tm, GC content, secondary structure)
2.3 Other Fluorescent Probe Technologies
| Probe Type | Principle | Advantages | Application Scenarios |
|---|---|---|---|
| Molecular Beacon | Hairpin structure; fluorescence restored upon target binding | High specificity, can distinguish single-base differences | SNP detection |
| Hybridization Probes (FRET) | Adjacent pair; energy transfer upon hybridization | Can be used for melting curve analysis | Genotyping |
| Eclipse Probe | Duplex-like structure; fluorescence enhanced upon hybridization | Low background | Multiplex detection |
| LNA Probe | Locked nucleic acid modification; higher Tm | Very high detection sensitivity | Rare mutation detection |
2.4 Passive Reference Dye (ROX)
ROX is an inert fluorescent dye that does not participate in PCR reactions. It is used to normalize fluorescence signal differences between wells.
ROX Functions:
-
Corrects for well-to-well volume differences
-
Corrects for volume changes due to evaporation
-
Corrects for well-to-well differences in optical detection systems
Instrument ROX Requirements:
| Instrument Brand | ROX Requirement | Explanation |
|---|---|---|
| Applied Biosystems | ROX required | Uses ROX for signal normalization |
| Bio-Rad | ROX not required | Different optical system design |
| Roche | ROX not required | No normalization needed |
| Agilent | ROX optional | Can be added or not |
| Qiagen | Instrument-dependent | Refer to manual |
III. Key Raw Materials and Solutions for qPCR
3.1 Hot-Start DNA Polymerase — The "Engine" of Amplification
qPCR has specific requirements for DNA polymerases: must have hot-start properties to prevent non-specific amplification and primer dimers; requires high processivity to support efficient amplification; typically requires 5'→3' exonuclease activity to support TaqMan probe cleavage.
| Enzyme Type | Features | Application Scenarios | Recommended Specification |
|---|---|---|---|
| Antibody-Modified Hot-Start Taq | Anti-Taq antibody inhibits activity; activated at 95°C | Routine qPCR | 5-10 U/μL |
| Chemically Modified Hot-Start Taq | Chemical group blocks activity; requires longer activation | High-sensitivity detection | 5-10 U/μL |
| Hot-Start Taq (5'→3' Exonuclease Activity) | Supports TaqMan probe cleavage | TaqMan qPCR | 5-10 U/μL |
| Engineered Hot-Start Polymerase | High processivity, inhibitor-tolerant | Complex sample detection | 5-10 U/μL |
Advantages of Hot-Start Taq in qPCR:
| Feature | Standard Taq | Hot-Start Taq |
|---|---|---|
| Non-Specific Amplification | High | Low |
| Primer Dimers | Common | Rare |
| Low-Copy Detection Ability | Low | High |
| Multiplex qPCR Suitability | Poor | Good |
3.2 Nucleotides (dNTPs, dUTP) — The "Building Blocks"
qPCR requires extremely high purity dNTPs (≥99% by HPLC), as impurities can affect fluorescence detection accuracy and amplification efficiency.
| Raw Material | CAS No. | Recommended Specification | Function |
|---|---|---|---|
| dATP | 1927-31-7 | ≥99% (HPLC), DNase/RNase free | Adenine nucleotide |
| dCTP | 2056-98-6 | ≥99% (HPLC), DNase/RNase free | Cytosine nucleotide |
| dGTP | 2564-35-4 | ≥99% (HPLC), DNase/RNase free | Guanine nucleotide |
| dTTP | 365-08-2 | ≥99% (HPLC), DNase/RNase free | Thymine nucleotide |
| dUTP | 1175-53-7 | ≥99% (HPLC), DNase/RNase free | Used with UNG for carryover prevention |
dNTP Considerations for qPCR:
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Use dUTP to replace part or all of dTTP along with UNG enzyme to prevent carryover contamination
-
Excess dNTP concentration can inhibit fluorescence signal (especially SYBR Green)
-
Poor quality dNTPs cause abnormal amplification curves (low plateau, Ct value drift)
3.3 Primers and Probes — The "Navigation System" of Detection
qPCR primer design is similar to traditional PCR but requires higher specificity and efficiency. Probe design is critical for successful TaqMan qPCR.
Special qPCR Primer Design Requirements:
| Parameter | Traditional PCR | qPCR | Explanation |
|---|---|---|---|
| Amplicon Length | 100-2000 bp | 70-150 bp | Shorter amplicons improve efficiency |
| Primer Tm | 50-65°C | 58-62°C | Match probe Tm |
| Tm Difference | ≤5°C | ≤2°C | Forward and reverse primers have similar Tm |
| Amplification Efficiency | Not critical | Must be 90-110% | Affects quantification accuracy |
TaqMan Probe Design Guidelines:
| Parameter | Recommended Range | Explanation |
|---|---|---|
| Length | 18-30 nt | Shorter than primers |
| Tm | 65-70°C | 5-10°C higher than primer Tm |
| GC Content | 40-65% | Avoid consecutive Gs |
| 5' End | Cannot be G | G quenches fluorescence |
| Relation to Primers | No overlap with primer annealing regions | Avoid interference |
| Secondary Structure | No hairpins or dimers | Affects hybridization efficiency |
3.4 Buffer and Mg²⁺ — The "Environment Stabilizers" of Amplification
qPCR buffer needs to provide optimal conditions for the polymerase while supporting fluorescence dye stability and probe hybridization efficiency.
Key qPCR Buffer Components:
| Component | Recommended Concentration | Function |
|---|---|---|
| Tris-HCl (pH 8.5-9.0) | 10-50 mM | Maintains pH stability |
| KCl | 20-50 mM | Provides ionic strength |
| (NH₄)₂SO₄ | 15-30 mM | Increases specificity (optional) |
| MgCl₂ | 2-6 mM | Polymerase cofactor |
| BSA | 0.1-1 mg/mL | Stabilizes enzyme, reduces adsorption |
| Glycerol | 5-10% | Protects enzyme activity |
| Tween-20 / Triton X-100 | 0.01-0.1% | Improves enzyme activity |
Importance of Mg²⁺ in qPCR:
-
Mg²⁺ concentration affects polymerase activity, primer annealing, and product specificity
-
For SYBR Green systems, Mg²⁺ concentration affects fluorescence signal intensity
-
For TaqMan systems, Mg²⁺ concentration affects probe cleavage efficiency
-
Optimal concentration must be optimized based on primers, probes, and template (typically 2-6 mM)
3.5 Passive Reference Dye (ROX)
| Feature | Explanation |
|---|---|
| Function | Normalizes well-to-well fluorescence differences, improves data accuracy |
| Applicable Instruments | Applied Biosystems (7300, 7500, 7900, QuantStudio series) |
| Recommended Concentration | Instrument-dependent: Low ROX (e.g., 7500) or High ROX (e.g., 7900) |
| Storage Conditions | -20°C, protect from light |
3.6 Stabilizers and Preservatives
| Raw Material | CAS No. | Recommended Concentration | Main Application | Precautions |
|---|---|---|---|---|
| BSA | 9048-46-8 | 0.1-1 mg/mL | Stabilizes enzyme, reduces adsorption | DNase/RNase free |
| Trehalose | 6138-23-4 | 2-5% | Lyoprotection, master mix thermal stability | Extends shelf life |
| Glycerol | 56-81-5 | 5-50% | Cryoprotection for enzymes | Store at -20°C |
| ProClin 300 | — | — | ⚠️ Prohibited in qPCR master mixes | Inhibits polymerase |
| Sodium azide | 26628-22-8 | — | ⚠️ Prohibited in qPCR master mixes | Inhibits polymerase |
| DMSO | 67-68-5 | 1-5% | Reduces secondary structure, improves amplification | For GC-rich templates |
Critical Warnings:
-
⚠️ qPCR reagents (including master mixes) must NOT contain sodium azide or ProClin 300 — both strongly inhibit DNA polymerase activity
-
⚠️ Fluorescent probes must be stored protected from light — photobleaching reduces fluorescence signal
-
⚠️ SYBR Green I must be stored protected from light — prolonged light exposure causes degradation
IV. qPCR Formulation Examples
4.1 2× qPCR Master Mix (SYBR Green) Formulation
| Component | Concentration (2×) | Final Concentration (1×) | Function |
|---|---|---|---|
| Tris-HCl (pH 8.5) | 40-100 mM | 20-50 mM | Buffer |
| KCl | 100 mM | 50 mM | Ionic strength |
| MgCl₂ | 4-8 mM | 2-4 mM | Cofactor |
| dNTPs each | 400-600 μM | 200-300 μM | Nucleotide substrates |
| Hot-Start Taq Polymerase | 2-5 U/μL | 1-2.5 U/μL | DNA amplification |
| SYBR Green I | 2-5× | 1-2.5× | Fluorescent dye |
| ROX (optional) | As appropriate | — | Passive reference dye |
| BSA | 1-2 mg/mL | 0.5-1 mg/mL | Stabilizer |
| Glycerol | 10% | 5% | Cryoprotectant |
| Stabilizers | As appropriate | — | Extends shelf life |
4.2 2× qPCR Master Mix (TaqMan Probe) Formulation
| Component | Concentration (2×) | Final Concentration (1×) | Function |
|---|---|---|---|
| Tris-HCl (pH 8.5) | 40-100 mM | 20-50 mM | Buffer |
| KCl / (NH₄)₂SO₄ | 100-160 mM | 50-80 mM | Ionic strength |
| MgCl₂ | 4-8 mM | 2-4 mM | Cofactor |
| dNTPs (with dUTP) | 400-600 μM | 200-300 μM | Nucleotide substrates |
| Hot-Start Taq Polymerase | 2-5 U/μL | 1-2.5 U/μL | DNA amplification + probe cleavage |
| UNG enzyme (optional) | 0.2-0.5 U/μL | 0.1-0.25 U/μL | Prevents carryover contamination |
| ROX (optional) | As appropriate | — | Passive reference dye |
| BSA | 1-2 mg/mL | 0.5-1 mg/mL | Stabilizer |
| Glycerol | 10% | 5% | Cryoprotectant |
4.3 Primer-Probe Mix Formulation (20×)
| Component | Final Concentration (1×) | Concentration (20× Stock) | Explanation |
|---|---|---|---|
| Forward Primer | 100-500 nM | 2-10 μM | Each primer final concentration |
| Reverse Primer | 100-500 nM | 2-10 μM | Each primer final concentration |
| TaqMan Probe | 50-250 nM | 1-5 μM | Protect from light |
| Tris-HCl (pH 8.0) | 10 mM | — | Buffer |
| EDTA | 0.1 mM | — | Chelating agent |
4.4 Standard qPCR Reaction System (20 μL)
| Component | Volume | Final Concentration | Explanation |
|---|---|---|---|
| 2× qPCR Master Mix | 10 μL | 1× | — |
| Primer-Probe Mix (20×) | 1 μL | 1× | If using |
| Forward Primer (10 μM) | 0.4-1 μL | 0.2-0.5 μM | If adding separately |
| Reverse Primer (10 μM) | 0.4-1 μL | 0.2-0.5 μM | If adding separately |
| Template DNA/cDNA | 2-5 μL | 1 pg - 100 ng | Adjust based on template type |
| Water (Molecular Biology Grade) | Bring to 20 μL | — | — |
4.5 Typical qPCR Thermal Cycling Program (Two-Step)
| Step | Temperature | Time | Cycles | Fluorescence Acquisition | Explanation |
|---|---|---|---|---|---|
| Initial Denaturation | 95°C | 2-10 minutes | 1 | No | Activate hot-start enzyme |
| Denaturation | 95°C | 10-20 seconds | 40 | No | Strand separation |
| Annealing/Extension | 60°C | 20-60 seconds | 40 | Yes | Primer annealing + extension + fluorescence collection |
| Melting Curve (SYBR Green) | 60-95°C | Stepwise increase | 1 | Yes | Differentiates non-specific products |
Special Requirements for TaqMan Programs:
-
Annealing/extension step must be long enough for probe binding and cleavage (typically ≥30 seconds)
-
Fluorescence acquisition is always at the end of the annealing/extension step
V. qPCR Data Analysis
5.1 Amplification Curve
| Phase | Explanation | Characteristics |
|---|---|---|
| Baseline Phase | Low fluorescence, not yet above background | Cycles 0-10 |
| Exponential Phase | Fluorescence begins exponential growth | Cycles 10-25, best quantification window |
| Linear Phase | Amplification efficiency decreases, signal plateaus | Cycles 25-30 |
| Plateau Phase | Product accumulation reaches saturation | Cycles >30 |
5.2 Melting Curve (SYBR Green)
Melting curve analysis is an important tool for verifying amplification specificity in SYBR Green method.
| Feature | Single Peak | Multiple Peaks |
|---|---|---|
| Meaning | Single amplification product | Multiple products or primer dimers |
| Melt Temperature (Tm) | Product-specific, correlated to GC content and length | — |
| Corrective Action | Can proceed with quantification | Need to optimize primers or annealing temperature |
5.3 Standard Curve
| Parameter | Meaning | Acceptance Criteria |
|---|---|---|
| R² (Coefficient of Determination) | Goodness of linear fit | ≥0.99 |
| Amplification Efficiency (E) | E = 10^{-1/slope} - 1 | 90-110% (1.9-2.1 fold/cycle) |
| Slope | Standard curve slope | -3.1 to -3.6 |
5.4 Absolute vs Relative Quantification
| Quantification Method | Calculation | Standards Required | Application Scenarios |
|---|---|---|---|
| Absolute Quantification | Standard curve → interpolate unknowns | Yes | Viral load, pathogen quantification |
| Relative Quantification (ΔΔCt Method) | ΔCt = Ct(target) - Ct(reference); ΔΔCt = ΔCt(treated) - ΔCt(control); Fold change = 2^{-ΔΔCt} | No | Gene expression analysis |
VI. Frequently Asked Questions (FAQ)
Q1: No amplification curve or Ct value too high in qPCR. What are possible causes?
A: No signal or high Ct values are usually related to the following factors:
| Cause | Solution |
|---|---|
| Insufficient or degraded template | Increase template amount, use fresh template |
| Primer design issue | Redesign primers, check Tm and amplicon length |
| Probe design issue (TaqMan) | Check probe Tm is 5-10°C higher than primer Tm, avoid 5' G |
| Reagent preparation issue | Check for missing components, use fresh reagents |
| Thermal cycling program issue | Check annealing temperature, ensure sufficient initial denaturation time |
| Instrument issue | Check fluorescence channel settings, confirm ROX is not interfering |
Q2: How to resolve non-specific amplification or primer dimers in qPCR?
A: Non-specific amplification is usually related to the following factors:
| Cause | Solution |
|---|---|
| Annealing temperature too low | Increase annealing temperature by 2-5°C |
| Mg²⁺ concentration too high | Reduce Mg²⁺ concentration by 0.5-1 mM |
| Primer concentration too high | Reduce primers to 100-200 nM |
| Too much template | Dilute template 10-100 fold |
| Too many cycles | Reduce cycles to 35-40 |
| Non-hot-start polymerase | Use hot-start Taq polymerase |
Q3: How to improve low amplification efficiency (<90%) in qPCR?
A: Low amplification efficiency is usually related to the following factors:
| Cause | Solution |
|---|---|
| Primer design issue | Optimize primers, ensure amplicon length 70-150 bp |
| Probe design issue (TaqMan) | Check probe Tm and secondary structure |
| Insufficient Mg²⁺ concentration | Increase Mg²⁺ concentration to 3-5 mM |
| Insufficient or excessive dNTP concentration | Optimize dNTP concentration (200-300 μM each) |
| Inhibitors in template | Dilute template, use inhibitor-tolerant polymerase |
| Suboptimal thermal cycling conditions | Check ramp rates, extend extension time |
| Degraded reagents | Use fresh reagents, check storage conditions |
Q4: How to resolve multiple peaks in melting curve (SYBR Green)?
A: Multiple melting curve peaks indicate non-specific products or primer dimers:
| Cause | Solution |
|---|---|
| Non-specific amplification | Increase annealing temperature, redesign primers |
| Primer dimers | Redesign primers (avoid complementary sequences) |
| Template contamination | Use nuclease-free water as negative control |
| Genomic DNA contamination (RNA samples) | Treat RNA samples with DNase I |
Q5: How to resolve poor qPCR reproducibility with high well-to-well Ct variation?
A: Poor reproducibility is usually related to the following factors:
| Cause | Solution |
|---|---|
| Inaccurate pipetting | Use master mix to reduce pipetting steps, calibrate pipettes |
| Evaporation | Use high-quality sealing film, ensure tight seal |
| Bubbles | Centrifuge after loading (1000 rpm, 1-2 minutes) |
| Poor plate quality | Use qPCR-specific plates |
| Instrument well-to-well variation | Use ROX passive reference dye for normalization (if applicable) |
| Reagents not mixed | Mix thoroughly before use |
Q6: Can sodium azide or ProClin 300 be used as preservatives in qPCR reagents?
A: Absolutely NOT.
-
Sodium azide: Strongly inhibits DNA polymerase activity
-
ProClin 300: Also inhibits DNA polymerase activity
Recommendation: Use molecular biology grade water and aseptic technique. For master mixes requiring stability, use trehalose (2-5%) and BSA (0.1-1 mg/mL) as stabilizers.
Q7: How to determine if qPCR amplification efficiency is acceptable?
A: Assess through standard curve analysis:
| Parameter | Acceptance Range | Formula |
|---|---|---|
| R² | ≥0.99 | Linear correlation coefficient |
| Amplification Efficiency (E) | 90-110% | E = (10^{-1/slope} - 1) × 100% |
| Slope | -3.1 to -3.6 | — |
Q8: How to choose between SYBR Green and TaqMan methods?
A: Choice depends on experimental purpose and budget:
| Consideration | SYBR Green | TaqMan |
|---|---|---|
| Cost | Low | High (probe synthesis required) |
| Specificity | Lower (requires melting curve verification) | High (extra hybridization specificity) |
| Multiplex Detection | Single channel | Multi-channel (up to 5-6 plex) |
| Suitable Scenarios | Primer screening, gene expression, R&D | Clinical diagnostics, viral load, multiplex detection |
| Experimental Time | Short (no probe design) | Longer (probe design and validation) |
Q9: What is the correct method for using ROX in qPCR?
A: Follow these principles:
| Step | Operation |
|---|---|
| Confirm if instrument requires ROX | Consult instrument manual |
| Use master mix recommended concentration | High ROX (e.g., Applied Biosystems 7900) or Low ROX (e.g., 7500) |
| Avoid ROX excess | Signal saturation affects normalization |
| Store ROX protected from light | Photobleaching reduces signal |
Q10: How to prevent carryover contamination in qPCR?
A: Follow these contamination control strategies:
| Measure | Operation |
|---|---|
| Physical Separation | Strictly separate sample prep, reaction setup, and amplification/detection areas |
| Use dUTP/UNG System | Use dUTP to replace part of dTTP in master mix; add UNG enzyme; degrade dU-containing contaminants during initial PCR step |
| Dedicated Pipettes | Use separate pipettes for each area |
| Use Filter Tips | Prevent aerosols from entering pipettes |
| Regular Cleaning | Wipe work surfaces with 10% bleach or PCR cleaner |
VII. Summary
qPCR, as the most core quantitative detection technology in molecular diagnostics, has reagent performance that heavily depends on the proper selection and combination of hot-start DNA polymerases, dNTPs, fluorescent probes/dyes, passive reference dyes (ROX), buffers, stabilizers, and other raw materials.
艳 李
