การใช้งาน PCR แบบเรียลไทม์ในคลินิก

Real-time PCR: Clinical Applications

• Real-time PCR is a specialized technique that visualizes a PCR reaction "in real time" as the reaction progresses.
• It links an optical system to a thermal cycler, capturing signals at each reaction step.
• Also known as quantitative Polymerase Chain Reaction (qPCR).

Learning Outcomes

• Understand the principles of real-time PCR.
• Understand how to interpret real-time PCR results.
• Understand how to apply real-time PCR in a clinical setting.

What is Real-Time PCR?

• A specialized technique visualizing PCR reactions in real-time.
• Links an optical system to a thermal cycler,
• Captures signals at each reaction step.

Fluorescence Signal Detection

• Dye-based:
  • Ethidium bromide (1st generation)
  • SYBR Green I (2nd generation)
  • LC Green Plus (3rd generation)
• Probe-based:
  • Molecular beacons
  • FRET
  • Scorpions
  • Hybridization
  • Hydrolysis (TaqMan, 5' nuclease assay, TaqMan MGB)

Differences between Methods

• Visual demonstration of the differences between SYBR Green, Hybridization Probes, and TaqMan Probes.

Real-Time PCR Result

• Shows a graph of Relative Fluorescence Units (RFU) versus the number of PCR cycles.
• Includes phases like initiation, exponential, linear, and plateau.
• Indicates Cycle threshold (Ct), baseline, threshold, and other key terms for analysis.
• Also includes melting point graph (End-run Dissociation Curve, melting curve analysis)

Types of Real-Time Quantification

• Absolute Quantification: Uses a standard curve generated from serially diluted standards of known concentrations to calculate the concentration of an unknown sample.
• Relative Quantification: Measures the target based on either an external standard or a reference sample using various methods like target/reference ratio, comparative Ct (2-ΔΔCt) method, Pfaffl model, or Q-Gene.

Clinical Applications of Real-Time PCR: Viral Infection

• Viral load has the strongest impact on virology.
• Factors contributing to this include viral genetic simplicity.
• Ease of nucleic acid isolation.
• Less effort required compared to culture methods for other pathogens.
• Also helps to identify co-infections.

Clinical Applications of Real-Time PCR: Bacterial Infection

• Preferred method for diagnosing bacteria difficult to cultivate.
• Includes methods for identifying pathogens such as Listeria monocytogenes, Legionella pneumophila, Mycobacterium spp., Propionibacterium spp., Borrelia burgdorferi, and Leptospira genospecies.
• Provides relevant information for physicians and veterinarians quickly to determine appropriate antibiotic usage for infections.
• Identifies and monitors antibiotic resistance in bacterial isolates such as S. aureus, S. epidermidis, Helicobacter pylori, and Enterococcus species.

Clinical Applications of Real-Time PCR: Cancer Diagnosis

• Used for expression analysis of genes and differentiation of gene polymorphisms.
• Identifies gene duplications, deletions, fusions, and overexpression.

Clinical Applications of Real-Time PCR: Gene Expression

• Used to analyze gene expression levels.
• Demonstrates difference in gene expression between conditions.

RT-PCR vs Real-Time PCR

• RT-PCR (Reverse Transcriptase PCR) is a different technique than real-time PCR.
• RT-PCR involves two steps: cDNA generation (using mRNA as a template) and cDNA amplification.
• Quantitative Real-Time PCR (qPCR) is a combination of PCR and real-time analysis which allows precise quantification of DNA.

Quantitative Reverse Transcription PCR (RT-qPCR)

• Uses reverse transcription to convert RNA into cDNA followed by amplification via real-time PCR to detect and quantify RNA or mRNA.

cDNA Synthesis

• Process of converting mRNA into cDNA.
• Uses different primers like oligo dT and random primers.
• A crucial step in several molecular biology applications, including RT-qPCR.

RT-PCR vs qPCR vs RT-qPCR

• Descriptions of each method, demonstrating the separate processes and steps involved.

Normalization

• Corrects for sample-to-sample variation affecting gene expression data.
• Uses housekeeping genes (mRNA), including GAPDH, β-actin, and 18S Ribosomal RNA (rRNA).

ΔΔCt Method

• A method for calculating the difference in cycle thresholds to determine the fold change in gene expression between treated and control groups.