Real-Time PCR & Pathogen Diagnostics Course

Lesson 1 • Pathogen Genome Organisation

Surveys bacterial, viral, fungal, and parasitic genome structures. Provides context for selecting diagnostic target regions.

Lesson 2 • Nucleic Acid Structure and Function

Covers DNA and RNA architecture, base pairing, and strand polarity. Establishes the structural logic underlying primer design and amplification.

Lesson 3 • Nucleic Acid Extraction Principles

Introduces cell lysis, nucleic acid isolation, and purification logic. Prepares students for hands-on extraction protocols in later chapters.

Lesson 1 • PCR Inhibitors and Troubleshooting

Identifies common inhibitors in clinical and environmental samples and their mitigation. Equips students to diagnose failed or weak PCR reactions.

Lesson 2 • Gel Electrophoresis for PCR Products

Covers agarose gel preparation, electrophoresis, and band interpretation. Provides the visualisation skill needed to confirm amplification results.

Lesson 3 • Thermocycling and Amplification Kinetics

Explains denaturation, annealing, and extension phases and their temperature logic. Links cycle parameters to product yield and fidelity.

Lesson 4 • Primer Design Principles

Teaches GC content, melting temperature, and secondary structure rules for primer design. Directly enables specific amplification of pathogen targets.

Lesson 1 • Nucleic Acid Quantification and Purity

Covers spectrophotometric, fluorometric, and electrophoretic quality assessment methods. Ensures extracted material meets input requirements for downstream PCR assays.

Lesson 2 • Manual Extraction Protocols

Teaches phenol-chloroform, silica spin column, and magnetic bead manual methods. Builds foundational extraction skills applicable across sample types.

Lesson 3 • RNA Extraction and cDNA Synthesis

Addresses RNase control, RNA-specific lysis, and reverse transcription for RT-PCR. Enables detection of RNA viruses and host gene expression targets.

Lesson 4 • Clinical Sample Types and Handling

Covers blood, swabs, urine, CSF, tissue, and stool as diagnostic matrices. Establishes pre-analytical handling rules that protect nucleic acid integrity.

Lesson 1 • Amplification Curves and Cq Values

Teaches baseline, exponential, and plateau phases of the amplification curve. Establishes Cq as the primary quantitative readout.

Lesson 2 • Real-Time PCR Instrument Platforms

Surveys optical systems, block formats, and multiplexing capabilities of major platforms. Prepares students to select instruments for specific diagnostic needs.

Lesson 3 • Assay Optimisation for Real-Time PCR

Covers primer and probe concentration titration, annealing temperature gradients, and efficiency testing. Produces validated, reproducible assay conditions.

Lesson 4 • Melt Curve Analysis

Explains post-amplification melt curve generation and interpretation. Enables discrimination of specific products from primer dimers and non-specific amplicons.

Lesson 1 • Digital PCR for Absolute Quantification

Introduces droplet and chip-based digital PCR partitioning and Poisson statistics. Provides a standard-free quantification alternative for low-copy targets.

Lesson 2 • Absolute Quantification Methods

Teaches standard curve construction using serial dilutions of known copy-number standards. Enables direct pathogen load determination from clinical samples.

Lesson 3 • Statistical Analysis of PCR Data

Covers variance, confidence intervals, and appropriate statistical tests for PCR datasets. Ensures defensible interpretation of quantitative diagnostic results.

Lesson 4 • Relative Quantification and Reference Genes

Explains delta-delta Cq method and reference gene selection criteria. Applies to gene expression studies and normalised pathogen load comparisons.

Lesson 1 • Bacterial Pathogen Assay Design

Applies target selection and primer design to common bacterial pathogens. Addresses 16S rRNA, species-specific genes, and virulence factor targets.

Lesson 2 • Fungal and Parasitic Assay Design

Addresses ITS regions, species-specific markers, and parasite-specific targets. Extends assay design skills to eukaryotic pathogen diagnostics.

Lesson 3 • Target Gene Selection Strategies

Teaches criteria for choosing conserved, unique, and clinically relevant genomic targets. Directly determines assay sensitivity, specificity, and diagnostic utility.

Lesson 4 • Viral Pathogen Assay Design

Covers RNA and DNA virus target selection, RT-PCR design, and viral load quantification. Addresses genome variability and strain coverage challenges.

Lesson 1 • Mechanisms of Antimicrobial Resistance

Reviews enzymatic inactivation, target modification, efflux, and permeability resistance mechanisms. Provides the biological basis for selecting resistance gene targets.

Lesson 2 • Mutation Detection and Genotyping

Teaches allelic discrimination, high-resolution melt, and sequencing-based mutation detection. Identifies point mutations conferring resistance or treatment failure.

Lesson 3 • PCR Detection of Resistance Genes

Covers primer design for resistance gene families and multiplex resistance panels. Enables rapid genotypic resistance profiling from clinical samples.

Lesson 1 • Laboratory Quality Management Systems

Covers document control, corrective action, and quality indicators for molecular labs. Connects quality system elements to diagnostic accuracy and accreditation.

Lesson 2 • Biosafety in Molecular Diagnostics

Reviews biosafety levels, personal protective equipment, and pathogen inactivation requirements. Ensures safe handling of infectious clinical specimens.

Lesson 3 • External Quality Assessment and Proficiency

Explains proficiency testing schemes, panel performance evaluation, and corrective response. Demonstrates ongoing laboratory competency to accreditation bodies.

Lesson 4 • Regulatory Frameworks for Diagnostic Assays

Covers in vitro diagnostic device classification, performance evaluation requirements, and laboratory-developed test oversight. Prepares students to navigate assay approval pathways.