Inhibition Controls for Qualitative Real-Time PCR Assays: Are They Necessary for All Specimen Matrices?

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR as-says determined the overall inhibition rate to be 0.87 % when the inhibition control was added preextraction to 5,613 speci-mens and 0.01 % when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibi-tion rates of<1 % were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue. Inhibitors can be found in various specimen matrices, andthese substances can interfere with PCRs by interacting di-rectly with DNA and blocking the activity of the polymerase or other PCR mixture components (e.g., MgCl2), thereby pre-venting target amplification. Examples of PCR inhibitors in-clude bile salts in feces, heme in blood, and urea in urine. In addition, some components of common laboratory collection devices (e.g., viral transport medium, heparin, formalin, or swabs containing gel or charcoal) are known inhibitors of PCR. Inhibition (or internal) controls added directly to the specimen are often used in order to detect inhibition associated with the specimen matrix or the processing method (1, 2). Clinical and Laboratory Standards Institute document MM3-A2 recom-mends that the addition of an inhibition control be determined on a case-by-case basis by specimen type and with consider-ation of the potential consequences of a false-negative result (3). The College of American Pathologists recommends spik-ing an aliquot of the clinical specimen with target nucleic acid but indicates that “the practice can be discontinued once the laboratory accumulates sufficient data that the inhibition rate falls within acceptable limits ” (MIC 63278