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Elevating PCR results

Molecular

In today’s cost-conscious healthcare environment, consistent and reliable results are crucial for laboratory success.

Despite being relatively straightforward, PCR testing is not impervious to error—missteps can result in poor sensitivity and/or specificity. Every step in the PCR process must be rigorously controlled and assessed, properly calculating and applying precise denaturation and annealing temperatures during cycling to determine appropriate primer and probe concentrations for your desired targets, and more.¹

Given the high degree of precision required to maintain robust laboratory processes, optimization is crucial to delivering reliable results. Labs that invest time and resources in a proper PCR setup are more likely to achieve reliable, reproducible, and robust results with higher specificity and sensitivity.

Maintaining result integrity

Accurate and reliable results lead to better outcomes all round, from medical research to patient diagnosis and care. Ensuring high-quality output requires precision throughout. Sample collection, transportation, sorting and preparation, storage, and QA procedures all need to be considered.

The quality and purity of samples is a key factor. Low-quality samples and contamination often compromise the end result, forcing labs to spend valuable resources correcting mistakes—this can include retraining laboratory and clinical staff (doctors, nurses etc.) Failure can be time-consuming and cost intensive. But with rigorous quality control and troubleshooting, you can produce highly accurate results every time.

To maintain result integrity and produce meaningful and reliable data at scale, there are certain performance checks which must take place.

Perform quality control during collection, transportation, and preparation.²

Use correct labelling and readable barcodes when sorting samples.

Ensure DNA/RNA are of the highest quality, including well-formulated reagents and properly designed primers and probes.

Prevent contamination by minimizing manual tasks with end-to end automation.

Today’s technology makes it possible to address these challenges and significantly improve the quality of PCR results. Combining Roche’s fully automated, high throughput analyzers with pre- and post-analytical solutions creates a fully automated workflow that offers more protection against costly errors, and unrivalled efficiency and speed.

References

Primer Digital. General Guidelines for PCR Optimization. https://primerdigital.com/pcr.html. Accessed 12 November 2020.
Labome. Assay Development: 5 Considerations and 8 Fundamentals. https://www.labome.com/method/How-to-Develop-Assays-5-Considerations-and-8-Fundamentals.html. Accessed 12 November 2020.

Key components for optimizing PCR

When it comes to PCR optimization, primers, probes, and the level of automation are of paramount importance.¹

Selecting the right primers and probes

Primer and probe designs have a significant influence on the success of PCR analyses,¹ and are critical for reliable and accurate quantification.

Primers and probes must be of the right quality and purity to meet the criteria necessary for optimization. The detection of specific targets requires well-designed primers and probes to ensure related non-target sequences do not compromise results.

Roche has continually innovated and developed our assay design for high sensitivity and specificity, to get the best PCR results.

The key is really to have the right primer and probe design in the right quality in a way that you can scale it up to manufacture in the quantities needed.

Matthias Hoppler
Senior Director, Assay Development - Molecular Diagnostics

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Watch Roche expert, Matthias Hoppler, discuss the role of reagents in optimizing PCR performance.

Roche’s commitment to advancing PCR

From the initial investment in PCR in the early 1990’s until today, Roche has been at the forefront of PCR progress. Explore Roche’s industry-defining innovations and see our vision for the future of this powerful technology.

References

Wadle S, Lehnert M, Rubenwolf S, Zengerle R, von Stetten F. Real-time PCR probe optimization using design of experiments approach. Biomol Detect Quantif. 2015;7:1-8.