Molecular Diagnostics
Main types and steps
Yaqoub Ashhab
Aptil 2025
Main Stages in Molecular Diagnostic Test Development
Number of candidates
Target
Identification Assay Design
and and
Biomarker Optimization
Discovery
Implementation
Analytical Validation Clinical Validation Regulatory Approval
and Scaling
Detection Dependent Stage Independent Interdependent
sensitivity limit Lorem Ipsum simply Stage Stage
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High penetrance variants from lab to bedside
Identification of potential
disease associated variants
Segregation Clinical
Clinical
validation
Number of variant
utility
Integration in
health care
Development phases
Low penetrance variants from lab to bedside
Identification of potential
disease associated variants
Number of variant
Replication
Clinical
validation
Clinical
utility
Integration in
health care
Development phases
Main Stages in Molecular Diagnostic Test Development
Number of candidates
Target
Identification Assay Design
and and
Biomarker Optimization
Discovery
Implementation
Analytical Validation Clinical Validation Regulatory Approval
and Scaling
Detection Dependent Stage Independent Interdependent
sensitivity limit Lorem Ipsum simply Stage Stage
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Technologies for detection and profiling of nucleic
acid sequence variations
Khodakov D et al.. Adv Drug Deliv Rev. 2016;105:3-19.
Factors Influencing Technique Selection
Objective of the Test:
• Screening
• Risk Assessment
• Diagnosis
Type of Molecule Targeted:
• DNA: PCR, NGS, and Sanger sequencing are commonly used.
• RNA: Reverse transcription PCR (RT-PCR) is effective for RNA viruses.
• Proteins: Techniques like mass spectrometry are used for protein-based diagnostics.
Accuracy and Sensitivity:
• High sensitivity is crucial for early detection (e.g., real-time PCR).
• Specificity ensures fewer false positives (e.g., FISH for chromosomal abnormalities).
Cost Considerations:
• Microarrays and NGS provide extensive data but are expensive.
• PCR offers a cost-effective solution for targeted diagnostics.
Turnaround Time:
• Rapid techniques like real-time PCR are preferred in urgent clinical settings.
Comparison of laboratory methods for direct detection
of infections
Example: Qualitative PCR vs. Quantitative PCR in microbiology
Qualitative PCR is widely used for:
• herpesviruses (HSV, VZV, CMV, EBV)
• Enteroviruses, Parvoviruses, Poxviruses
• Respiratory viruses, SARSCoV
• Neisseria meningitidis, Streptococcus pneumoniae, Bordetella
pertussis, and Borrelia spp.
Quantitative PCR is widely used for:
• CMV, EBV, HBV, HCV, and HIV.
Quantification provides information on the progression and prognosis
of disease, and effectiveness of antiviral treatment.
Methodologies for analysis of polymerase chain reaction
(PCR) products
Main Stages in Molecular Diagnostic Test Development
Number of candidates
Target
Identification Assay Design
and and
Biomarker Optimization
Discovery
Implementation
Analytical Validation Clinical Validation Regulatory Approval
and Scaling
Detection Dependent Stage Independent Interdependent
sensitivity limit Lorem Ipsum simply Stage Stage
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Analytical validity
It refers to the ability of the test to accurately and reliably measure the analyte (e.g., DNA, RNA, protein) of interest.
Aspect Definition Example
Sensitivity Ability to detect true positives Detecting low-frequency mutations in ctDNA
Specificity Ability to avoid false positives Avoiding cross-reactivity with non-target pathogens
Accuracy Closeness of results to the true value Correctly reporting variant allele frequency in NGS
Precision Consistency of results (repeatability/reproducibility) Consistent Ct values in qPCR
Lowest concentration of the analyte that can be reliably
Limit of Detection detected Detecting 0.1% mutant allele frequency in liquid biopsy
Linearity Proportionality of results to analyte concentration Linear relationship in viral load quantification
Consistent performance despite slight temperature
Robustness Resistance to small variations in experimental conditions changes
Interference Impact of non-target substances on test performance Avoiding inhibition by bile salts in stool-based PCR
Reportable Range Range of analyte concentrations where results are reliable Glucose meter range of 20–600 mg/dL
Reference
Standards Use of standardized materials for validation Aligning with WHO standards for antibody testing
Stability Performance consistency over time Reagents maintaining performance until expiration
Cross-Reactivity Avoidance of unintended detection of similar targets Avoiding detection of NTM in TB testing
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