Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories

Abstract

This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories

“…Suitability testing is performed in addition to the verification required for introduction of a new assay to the repertoire of tests for a laboratory. 55,73,89…”

“…Selection of the extraction process is critical in PCR assay development, with the extraction and PCR assay linked through concurrent validation as a system. 73 Recommended controls when performing extractions are listed in Table 1. An internal control is highly recommended and is discussed at length in an accompanying article.…”

“…Quantification is possible with rtPCR and RT-rtPCR; however, most VDLs do not routinely perform quantitative rtPCR assays. The absolute quantification of nucleic acid targets requires standard curves, and although necessary during development and validation, 73 standard curves are rarely used in routine veterinary diagnostic testing because of expense. Instead, the rtPCR cycle at which a sample is labeled positive (cycle threshold, Ct) is established based on the relative amount of target nucleic acid in the test sample in relation to that of a thoroughly characterized positive control sample.…”

“…Laboratories using pooling protocols should be aware that often the pooling potential is predicted from the Ct level of individual samples and is not empirically derived, and such predictions do not take into account the increased possibility of more inhibitors given the diversity and complexity of biological samples. A method comparability study 55,73,89 is appropriate before adopting a pooling strategy.…”

Focus issue on best practices for development, validation, and use of PCR assays

“…Among the topics specifically addressed in the focus issue papers are: a comparison of different international validation guidelines, with suggested practices for assay development and validation for real-time PCR assays 5 ; performance of pre-analytical, analytical, and post-analytical steps to ensure reliable real-time PCR assays 4 ; the different types of internal assay controls that would ideally be included in PCR-based testing to detect and manage the inhibitory substances commonly associated with different specimen matrices 6 ; guidance for sample preparation, submission to sequencing applications, and quality assessment of data obtained when using Sanger-based genetic sequencing in the context of diagnostic laboratory use 1 ; a methodologic review of test validation studies for infectious diseases in wild mammals with a focus on study design, statistical analysis, and reporting of results 2 ; and the report of a new open-source real-time PCR assay for Mycoplasma cynos using principles and guidance as outlined in the associated papers from the Laboratory Technology Committee. 3 The complete protocol of the M. cynos tuf assay is provided to facilitate assay harmonization.…”