Inhibition monitoring in veterinary molecular testing

Abstract

Many of the sample matrices typically used for veterinary molecular testing contain inhibitory factors that can potentially reduce analytic sensitivity or produce false-negative results by masking the signal produced by the nucleic acid target. Inclusion of internal controls in PCR-based assays is a valuable strategy not only for monitoring for PCR inhibitors, but also for monitoring nucleic acid extraction efficiency, and for identifying technology errors that may interfere with the ability of an assay to detect the intended target. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians reviewed the different types of internal controls related to monitoring inhibition of PCR-based assays, and provides information here to encourage veterinary diagnostic laboratories to incorporate PCR internal control strategies as a routine quality management component of their molecular testing.

Keywords: PCR inhibition; internal control; quality assurance; real-time PCR; veterinary molecular tests.

Figure 1.

Example of a properly titrated exogenous internal control (XIC), provided by the Laboratory Technology Committee of the AAVLD. The XIC (open circles) maintains a consistent cycle threshold (Ct) value over a wide target concentration range without compromising the limit of detection of the assay. Serial dilutions of equine herpesvirus 1 cell culture lysates were tested with a DNA XIC spiked into lysis buffer. Four replicates were tested at each viral concentration.

Figure 2.

Flow chart for common inhibition monitoring strategies for PCR. The test tube symbols represent the stage at which specific controls or reagents are added. EIC = endogenous internal control; IC = internal control; PAC = positive amplification control; PEC = positive extraction control; XIC = exogenous internal control. Strategy A outlines an approach in which XICs are added to lysis buffer in the initial step of the extraction and a PAC is incorporated at the PCR stage to monitor inhibition, extraction efficiency, and amplification. In strategy B, an assay-specific PEC is used with an XIC spiked at the PCR setup stage for monitoring all stages of the assay. Strategy C utilizes an EIC intrinsic to the sample that is quantified during amplification to monitor inhibition. Note that this chart does not cover negative controls, which should be incorporated at the extraction and setup stages.