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

Toohey-Kurth, Kathy; Mulrooney, Donna M.; Hinkley, Susanne; Killian, Mary Lea; Pedersen, Janice C.; Bounpheng, Mangkey A.; Pogranichniy, Roman M.; Bolin, Steve; Maes, Roger K.; Tallmadge, Rebecca L.; Goodman, Laura B.; Crossley, Beate

doi:10.1177/1040638720962076

Cited by 9 publications

(9 citation statements)

References 78 publications

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“…All controls are reviewed in companion articles in this focus issue. 44,58 Establishment of criteria to monitor assay performance is needed to complete the OIE validation pathway and to meet the AAVLD accreditation requirement. 2 In addition to daily monitoring of the assay, primer and probe sequences should be monitored routinely to determine whether emerging/novel nucleotide sequence mutations in the target region alter the performance of the assay.…”

Section: De Novo Validationmentioning

confidence: 99%

“…Once the target gene is selected, additional in silico evaluation of the primer and probe sequences is performed using commercial or publicly accessible software. 15,41,44 It is critical to perform a search of nucleotide databanks (https:// blast.ncbi.nlm.nih.gov) to determine if target sequences have homology to other agents in addition to the expected target. Design and selection of multiple primer/probe pairs for each target are advised so that the best choice can be made following empirical testing.…”

Section: Developmentmentioning

confidence: 99%

“…Additional recommended controls for routine performance of PCR are discussed in accompanying articles found in this focus issue. 44,58…”

Section: De Novo Development Of Rtpcrmentioning

confidence: 99%

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

Toohey-Kurth

Reising²,

Tallmadge

et al. 2020

J VET Diagn Invest

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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.

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Section: De Novo Validationmentioning

confidence: 99%

Section: Developmentmentioning

confidence: 99%

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

Toohey-Kurth

Reising²,

Tallmadge

et al. 2020

J VET Diagn Invest

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“…3 Technical advances in molecular diagnostics have overcome some of these limitations by using closedtubes assays and incorporating quality controls. 85 In experimental studies with both nasal secretions and blood available for qPCR testing, detection of EHV-1 in nasal secretions and blood ranged from An important limitation here is our use of a narrative review approach to evaluate the literature, which incorporated many elements of a systematic review process. The main difference between a narrative and a systematic review relates to the absence of an evaluation of the study quality of individual studies.…”

Section: Discussionmentioning

confidence: 99%

Viremia and nasal shedding for the diagnosis of equine herpesvirus‐1 infection in domesticated horses

Pusterla,

Dorman,

Burgess

et al. 2023

Veterinary Internal Medicne

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BackgroundEquine herpesvirus type 1 (EHV‐1) infection is associated with upper respiratory disease, EHM, abortions, and neonatal death.Research QuestionsAre nasal secretions a more sensitive biological sample compared to blood for the detection of EHV‐1 infection? How long is EHV‐1 detectable after primary infection by PCR?MethodsMedLine and Web of Science searches identified original peer‐reviewed reports evaluating nasal shedding and viremia using virus isolation methods or PCR published in English before October 9, 2023.ResultsSixty experimental and 20 observational studies met inclusion criteria. EHV‐1 detection frequency by qPCR in nasal secretions and blood from naturally‐infected horses with fever and respiratory signs were 15% and 9%, respectively; qPCR detection rates in nasal secretions and blood from horses with suspected EHM were 94% and 70%, respectively. In experimental studies the sensitivity of qPCR matched or exceeded that seen for virus isolation from either nasal secretions or blood. Detection of nasal shedding typically occurred within 2 days after EHV‐1 inoculation with a detection period of 3 to 7 days. Viremia lasted 2 to 7 days and was usually detected ≥1 days after positive identification of EHV‐1 in nasal secretions. Nasal shedding and viremia decreased over time and remained detectable in some horses for several weeks after inoculation.Conclusions and Clinical ImportanceUnder experimental conditions, blood and nasal secretions have similar sensitivity for the detection of EHV‐1 when horses are sampled on multiple consecutive days. In contrast, in observational studies detection of EHV‐1 in nasal secretions was consistently more successful.

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“…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.…”

mentioning

confidence: 99%

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

2020

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When PCR was first introduced for routine use in diagnostic laboratories in the 1980s, the technology opened the door to a new and dynamic future for detection of disease agents. However, the implementation of PCR technology in animal health laboratories far outpaced the development of documented guidelines and standards for use. In 2011, an effort was initiated within the American Association of Veterinary Laboratory Diagnosticians (AAVLD) membership to share PCR-based testing experiences more formally, as well as to document relevant laboratory science for the benefit of all animal health laboratories. The AAVLD "Laboratory Technology Committee" was formed during the annual 2011 AAVLD/USAHA meeting to provide a platform for laboratorians to exchange and discuss new testing technologies, including but not limited to molecular-based tests. The committee members devised a plan to combine the plethora of PCR-based testing experiences and best practices into general guidance documents to assist laboratories in establishing and performing PCR within their own unique laboratory settings. The committee formulated an overall goal of helping to provide consistent, high-quality molecular-based testing for the veterinary diagnostic laboratory community. This issue of the Journal of Veterinary Diagnostic Investigation, the Focus issue on PCR best practices, includes a series of 6 papers that represent the cumulative work of the committee over the ensuing years, as collated by a core group of primary authors. Over the years, members rotated off the committee, refocused their areas of interest, or retired from diagnostic work, while new diagnosticians with new experiences joined the ongoing effort. It is important for all of those individuals to know that the core authors of these focus issue papers have made a concerted effort to include all contributors to the relevant manuscripts as coauthors. To anyone who has been inadvertently missed, our sincere apologies and genuine thanks for your efforts.

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

Cited by 9 publications

References 78 publications

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

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

Viremia and nasal shedding for the diagnosis of equine herpesvirus‐1 infection in domesticated horses

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

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