Transferring a Quantitative Molecular Diagnostic Test to Multiple Real-Time Quantitative PCR Platforms

Area of Science:

• Molecular Biology
• Biotechnology
• Clinical Diagnostics

Background:

• Quantitative gene expression assays are vital for diagnosis and research.
• Assay performance is often limited to specific real-time quantitative PCR (qPCR) instrumentation.
• Lack of platform interoperability restricts the widespread application of established qPCR assays.

Purpose of the Study:

• To develop and validate a technical and statistical framework for transferring qPCR assays across different platforms.
• To ensure analytical and clinical validity is maintained during assay transfer.
• To enable broader operability of quantitative diagnostic tests.

Main Methods:

• A framework was developed for transferring established real-time qPCR assays.
• The MammaTyper assay was used to test feasibility across CFX96, Applied Biosystems 7500 Fast, and Mx3000P platforms, using LightCycler 480 II as a reference.
• Equivalence testing (Bland-Altman, Deming regression), method comparison for cutoff adjustment, and precision estimation were performed.

Main Results:

• Candidate platforms demonstrated high comparability to the reference, with a 7 log quantification range and 97%-103% amplification efficiencies.
• Equivalence tests successfully prequalified instruments, preventing errors and enabling reliable cutoff adjustments.
• Cross-platform agreement for markers and subtypes ranged from 91% to 100%, with kappa values between 0.78 and 1.00.

Conclusions:

• The proposed framework enables reliable transfer of real-time qPCR assays across different platforms.
• Platform-specific adjustments are crucial for maintaining assay performance and ensuring accurate results.
• This approach expands the utility of quantitative diagnostic tests without compromising validity.