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

The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.

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

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.

Detection of the parasitic nematode, Pseudocapillaria tomentosa, in zebrafish tissues and environmental DNA in research aquaria.

Although zebrafish continue to increase in popularity as a vertebrate animal model for biomedical research, chronic infectious diseases in laboratory populations remain prevalent. The presence of pathogens such as Pseudocapillaria tomentosa, a parasitic nematode found in the intestine of infected zebrafish, can significantly influence experimental endpoints and negatively impact reproducibility of research findings. Thus, there is a need for screening tests for zebrafish with the sensitivity to detect even low levels of pathogens present in tissues. Assays based on the detection of DNA are commonly used for such screening tests. Newer technologies such as digital PCR provide an opportunity to improve the sensitivity and precision of these assays, so they can be reliably used to detect pathogen DNA in water, reducing the need for lethal testing. We have designed a qPCR-based assay with the sensitivity to detect less than 5 copies of the P. tomentosa SSU-rDNA gene in tissues of infected zebrafish and environmental DNA from aquarium water housing infected fish. In addition, we adapted this test to a dPCR platform to provide a precise quantification of P. tomentosa DNA and demonstrate the resistance of this assay to inhibitors commonly found in freshwater aquaria.

Development of Quantitative Real-Time PCR Assays for Postmortem Detection of Mycobacterium spp. Common in Zebrafish (Danio rerio) Research Colonies.

Mycobacterium spp. infections are common in zebrafish kept in research facilities. These comorbidities can substantially modulate the responses of these fish to external and internal stimuli. Therefore, diagnostic tests to detect Mycobacterium spp. infections in zebrafish colonies prove essential. Here, we outline the development of quantitative simplex real-time PCR assays to detect the 3 Mycobacterium species most commonly identified in laboratory zebrafish. The assays targeted the heatshock protein 65 gene of M. marinum, M. chelonae, and M. haemophilum. The assays are both highly specific and sensitive for fresh-frozen samples and highly specific and moderately sensitive for formalin-fixed paraffin-embedded (FFPE) samples. Two sampling techniques for FFPE samples of sagittally sectioned zebrafish were evaluated. Both paraffin cores targeting granulomas containing bacteria and scrolls from the entire fish yielded DNA of equivalent quantity and purity. The diagnostic sensitivity of cores was superior to that of scrolls for M. chelonae and M. haemophilum but not M. marinum. The assays are cost-effective and ideally suited to diagnosing common Mycobacterium spp. infections in zebrafish.