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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Analytical Sensitivity and Specificity

Analytical sensitivity:
The assay’s ability to detect very low concentrations of a given substance in a biological specimen. Analytical sensitivity is often referred to as the limit of detection (LoD). LoD is the actual concentration of an analyte in a specimen that can be consistently detected ≥ 95% of the time. The LoD may be represented as the number of genome copies, infectious dose, colony-forming units, plaque forming units, etc. of the analyte that can be detected. To determine analytical sensitivity, an end-point dilution is used until the assay can no longer detect the target in question in more than 95% of the replicates. Analytical sensitivity can vary substantially for the same assay when different sample matrices are used. When setting up a dilution series, it is important to use a diluent that has qualities that are similar to the sample matrix (eg, blood, CSF).
Analytical specificity:
The assay’s ability to detect the intended target. It is critical to verify that the assay’s primers are specific to the target. There are two components to analytical specificity, cross reactivity and interference, discussed below.
  1. Cross reactivity may occur when genetically related organisms are present in a patient specimen. These organisms mimic the intended target, which results in the assay’s primers cross reacting or annealing to these genetically related organisms. For example, a nasopharyngeal specimen that is collected for Bordetella pertussis may contain normal respiratory flora along with other potential respiratory pathogens, such as Bordetella bronchiseptica. If the specimen is to be tested for B. pertussis, and the assay primers are not specific to this target, then the primers may anneal to other organisms which will generate a false positive result.
  2. Interference may occur when a specimen is introduced to endogenous substances like hemoglobin, bilirubin, medications, etc. or exogenous inhibitory substances, such as hand cream, powdered gloves, serum separators, etc. These substances will inhibit the primers from binding to the intended target. If the target amplifies without the primers attaching, the target will not be detected, potentially generating a false negative result.