Molecular Assay Design

Molecular Assay Design

  • Print

Results Driven by Sound Science

Viracor Eurofins set the standard in molecular infectious disease testing through innovative assay development. As leaders in infectious disease testing, we were one of the first to commercially offer quantitative, real-time PCR(qPCR) assays for adenovirus, BK virus(BKV), and JC virus(JCV), among others. We immerse ourselves in the clinical science of infectious diseases through continuous research, partnerships, and collaborations that translates into innovative assay designs. Bringing you the best results start with sound science.

Designing Multiple-Target Assays to Eliminate False Negative Results

The highly mutable nature of pathogens often proves challenging in PCR primer and probe design. Typically, qPCR assays are designed so that a  primer and probe set targets a single location within the pathogen genome. However, these single-target assays often prove inadequate when genetic mutations arise. Sequence-polymorphisms in the assay target region can cause impaired primer or probe binding, which may result in significant under-quantification or a false negative result. To eliminate this possibility, Viracor Eurofins qPCR assays often target two regions within the pathogen genome, utilizing unique primer and probe sets for each target site. The driving principle behind our multiple-target assay design is that when a mutation occurs in one assay target, the additional sets of primers and probes for the alternate target regions will still detect and accurately quantify the pathogen, and thus reduces the likelihood of under-qualification or a false negative result.

Viral Surveillance and Assay Refinement

At Viracor Eurofins, we believe that ensuring the accuracy of each diagnostic test is just as important as developing new ones, so we make pathogen surveillance and assay refinement a top priority. By utilizing nucleic acid sequence databases, each qPCR assay is frequently assessed to verify that new pathogen strains or genetic mutations that arise will not lead to a false negative result. If a new pathogen strain or genetic mutation is identified that would lead to a false-negative result, our current  qPCR assay is refined and revalidated to ensure that accurate test results are always reported. Continue to the Surveillance and Assay Refinement page for more details.

Increasing Sensitivity by Optimized Extraction Methods

We pay as careful attention to our extraction methodology as we do to primer and probe design. We understand that the sensitivity of PCR relies heavily on the efficiency of nucleic acid extraction. Therefore, we don't employ a "one size fits all" mentality. Our extraction methods are specimen-specific, ensuring maximum nucleic acid yields, to provide the most efficient, reproducible, and sensitive results.

Designing Assays to Account for All Strains

Viracor Eurofins designs each of our assays to account for all strains, serotypes, and clinical isolates - not just the most common ones. This is especially beneficial for immunocompromised patients who may be more likely to be infected with multiple or unusual strains. For example, the Viracor Eurofins Adenovirus qPCR assay detects all 51 serotypes with equal sensitivity. Additionally, Viracor Eurofins has designed and validated each assay to ensure there is no cross reactivity with other commonly occuring pathogens. This is critically important when testing for genetically similar but clinically divergent pathogens, such as BK virus and JC virus.

Increasing Clinical Relevance with Wide, Dynamic Assay Ranges

Viracor Eurofins designs our assays with the patient's needs in mind, so we design our assays so that they can provide wide reportable assay ranges. Our extremely high upper end ranges are critically important for viral pathogens such as BK virus, where clinically relevant values have been shown to begin at 1x107 copies/mL.2 Our exceptional low end sensitivity is invaluable for viral pathogens such as CMV, EBV, HHV-6, HBV, and HCV where very low viral loads are clinically

Ensuring Assay Consistency with Low Between-Run Variation

When you receive a result, you want to be confident that the result is reflecting a change in the patient's clinical condition, not a change due to variability of the assay over time. That is why low between run variability is critical when monitoring patients' viral load over an extended timeframe.

Quality and Reliability

The best assay design won't yield quality results if the assay isn't performed with a high degree of accuracy and consistency. At Viracor Eurofins, multiple assay controls and built-in redundancy and review processes are used at every step to ensure that physicians receive the most reliable and highest quality results.

- Full-process internal controls are included with each clinical specimen to eliminate false-negative results by serving to confirm that the extraction process was performed correctly and alerting for the presence of PCR inhibitors.
- Full-process positive and negative extraction controls as well as no template controls are included within each qPCR run to provide additional accuracy checks.


1. Tsai DE, Douglas L, Andreadis C, Vogl DT, Arnoldi S, Kotloff R, et al . EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: Results of a two-arm prospective trial. American Journal of Transplantation. 2008;8(5):1016-1024.

2. Pang XL, Doucette K, LeBlanc B, Cockfield SM, Preiksaitis JK. Monitoring of polyomavirus BK virus viruria and viremia in renal allograft recipients by use of a quantitative real-time PCR assay: one year prospective study. J Clin Micro. 2007;45(11):3568-3573.