IDEXX BioAnalytics uses hydrolysis probe-based real-time qPCR for all infectious agent testing. These assays are highly reproducible, specific for the intended target, and detect ≤10 template copies per PCR reaction. The PCR process takes the sample through multiple repetitive cycles of heating and cooling (thermocycles or cycles).  If the target sequence (template) for the PCR assay is present in the sample, the target-specific PCR product is exponentially amplified, and the probe is hydrolyzed generating a fluorescent signal for each cycle. The crossing point (Cp) or cycle threshold (Ct) is the cycle at which the cumulative fluorescence of the test reaches a detectable threshold; a number usually between 5 and 40.  In the validation process of real-time PCR assays, a dilution series of known concentrations of target template are tested and the template copy number per PCR reaction is plotted vs. the Cp values generated. This standard curve allows the Cp values generated for positive results to be mathematically converted into estimated template copy numbers per PCR reaction for positive results. When more template is present in the PCR reaction the fluorescent signal is detected sooner, resulting in a lower Cp value and greater estimated copy number per PCR reaction.

Multiple factors influence the amount of template present in samples collected for PCR analysis and in turn influence the estimated copy numbers per PCR reaction detected. These include, but are not limited to, the stage of infection, the number of copies of the target gene per infectious agent genome, the biology of the infectious agent, the biology of the host, the sample type tested, method of sample collection, sample storage, amount of sample submitted, and sample dilution or pooling. As such, samples testing positive by real-time PCR for infectious agents can have a range of copy numbers from single digits to millions of copies. For example, low copy numbers are seen commonly with positive results from environmental samples while cell lines positive for Mycoplasma spp. often contain high copy numbers. Considering the estimated copy number per PCR reaction can provide nuanced information to help interpret results in certain circumstances. For example, when using PCR to test animals after treatment for an infectious agent, if serial test results are positive with decreasing copy numbers, it may suggest that the treatment is working. We encourage you to use the reported copy numbers for informational purposes only and to not overinterpret the data. 

If you have questions about unexpected or unusual results, please follow up by promptly contacting the lab at idexxbioanalytics@idexx.com and discussing appropriate strategies for retesting the sample in question or by testing additional samples by PCR or alternative test methods.