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.

Download Full-text

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

Download Full-text

Validation of iQ-Check E. coli O157:H7 Real-Time PCR Test Kit for Detection of Escherichia coli O157:H7 in Selected Foods

Journal of AOAC International ◽

10.1093/jaoac/92.4.1095 ◽

2009 ◽

Vol 92 (4) ◽

pp. 1095-1104 ◽

Cited By ~ 1

Author(s):

Wendy F Lauer ◽

Sylvie Tymciu ◽

Caroline D Sidi ◽

Pierre Sonigo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Ground Beef ◽

Reference Method ◽

Target Sequence ◽

Apple Cider ◽

E Coli ◽

Test Kit ◽

Significant Difference ◽

The U.S

Abstract iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

Download Full-text

Evaluation of a Multiplex Real-Time PCR Method for Detecting Shiga Toxin–Producing Escherichia coli in Beef and Comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Method†

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-248 ◽

2014 ◽

Vol 77 (2) ◽

pp. 180-188 ◽

Cited By ~ 28

Author(s):

PINA M. FRATAMICO ◽

JAMIE L. WASILENKO ◽

BRADLEY GARMAN ◽

DANIEL R. DeMARCO ◽

STEPHEN VARKEY ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Virulence Genes ◽

Microbiology Laboratory ◽

Pcr Assay ◽

Department Of Agriculture ◽

E Coli ◽

System Method ◽

Inspection Service ◽

The U.S

The “top-six” non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 103 CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted for additional multiplex assays should regulations expand to include other O serogroups or virulence genes.

Download Full-text

Corrigendum

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638717728263 ◽

2017 ◽

Vol 29 (5) ◽

pp. 774-774

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Animal Health ◽

Pcr Assay ◽

Viral Dna ◽

University Of Florida ◽

Federal Research ◽

The University ◽

Swine Herds ◽

Domestic Swine

Sayler KA, Bigelow T, Koster LG, Swenson S, Bounds C, Hernández F, Wisely SM. Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds. J Vet Diagn Invest 2017;29:522–528. (Original doi:10.1177/1040638717706593). In the article titled “Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds” by Katherine A. Sayler et al., the Acknowledgements section, should read as follows: We thank all of the researchers who provided isolates or DNA samples of PRV, including C Romero at the University of Florida, College of Veterinary Medicine (U.S.); A Moreno at the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (Italy); S Verpoest at the CODA-CERVA Veterinary and Agrochemical Research Center (Belgium); A Steinrigl at the Austrian Agency for Health and Food Safety GmbH, Institute for Veterinary Disease Control (Austria); Z Dirbakova at the State Veterinary and Food Institute, Veterinary Institute in Zvolen (Slovakia); T Müller at the Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health (Germany); and F Ruiz-Fons and D González-Barrio at the Instituto de Investigación en Recursos Cinegéticos (IREC) (Spain).

Download Full-text

DEVELOPMENT OF SYBR GREEN-BASED REAL-TIME PCR FOR THE DETECTION OF CANINE, FELINE AND PORCINE PARVOVIRUSES

Taiwan Veterinary Journal ◽

10.1142/s1682648514500012 ◽

2014 ◽

Vol 40 (01) ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Chao-Nan Lin ◽

Chi-Hsien Chien ◽

Ming-Tang Chiou ◽

Jou-Wei Wang ◽

Ya-Ling Lin ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Simultaneous Detection ◽

Sybr Green ◽

Porcine Parvovirus ◽

Diagnostic Laboratory ◽

Vp2 Gene ◽

Coefficients Of Variation ◽

Causes Of Mortality

Parvovirus is now considered to be one of the most important infectious agents affecting canine, feline and porcine domestic animals. Canine parvovirus 2 (CPV 2) and feline parvovirus (FPV) are major infectious causes of mortality in puppies and kittens. Both CPV 2 and FPV have recently been shown to infect both dogs and cats. The characteristic symptoms of canine, feline and porcine parvovirus disease are intestinal hemorrhage with severe bloody diarrhea, leukopenia and reproductive failure, respectively. In this work, we describe the development of a novel real-time PCR system that is based on the use of SYBR Green and that allows the simultaneous detection of VP2 gene of CPV, FPV and porcine parvovirus (PPV). This system yielded low coefficients of variation for intra-assay and inter-assay variabilities. This novel SYBR Green-based real-time PCR assay was sensitive, specific and reliable for the amplification of CPV 2, FPV and PPV DNA, with a reproducible limit of detection of as few as 10 copies/μL of target DNA per reaction. The methods described in this study have been used successfully in our veterinary diagnostic laboratory and have been shown to be helpful tools for the diagnosis and quantification of parvovirus infection in canines, felines and swine.

Download Full-text

Differentiation of highly pathogenic strain H5N1 of AIV by Real Time PCR in the first outbreak in Romania

Biotechnology in Animal Husbandry ◽

10.2298/bah0701411z ◽

2007 ◽

Vol 23 (5-6-1) ◽

pp. 411-420

Author(s):

M. Zaulet ◽

S.E. Georgescu ◽

H. Coste

Keyword(s):

Avian Influenza ◽

Real Time ◽

Real Time Pcr ◽

Matrix Protein ◽

Animal Health ◽

Highly Pathogenic ◽

Pcr Screening ◽

Molecular Tests ◽

Poultry Farms ◽

Commercial Poultry

Fast diagnosis of Avian Influenza is a prerequisite for confining outbreaks. Diagnosis implies the differentiation of virulent and non-virulent Avian Influenza virus. After starting with PCR screening for matrix protein followed by identifying the presence of H5 and N1 genes, the diagnosis methodology within Romanian Institute for Diagnosis and Animal Health has moved to rapid molecular tests for detecting the virulent and nonvirulent strains. During October-December 2005 over 3400 biological specimens from 16 affected poultry backyards from Eastern Romania were tested by Real- Time PCR in the first Romanian outbreak. Over 3000 specimens have been tested in 2006, in the second Romanian outbreak when commercial poultry farms from central and southern Romania were affected.

Download Full-text

Surveillance for Avibacterium paragallinarum in autopsy cases of birds from small chicken flocks using a real-time PCR assay

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719844297 ◽

2019 ◽

Vol 31 (3) ◽

pp. 364-367 ◽

Cited By ~ 1

Author(s):

Kristin A. Clothier ◽

Andrea Torain ◽

Steve Reinl

Keyword(s):

Real Time ◽

Respiratory Disease ◽

Real Time Pcr ◽

Animal Health ◽

Delayed Diagnosis ◽

Pathogen Transmission ◽

Economic Losses ◽

Laboratory System ◽

Infectious Laryngotracheitis Virus ◽

Avibacterium Paragallinarum

Infectious coryza is a severe respiratory disease of chickens associated with large economic losses in affected commercial flocks. The fastidious causative pathogen, Avibacterium paragallinarum, is difficult to recover and identify, resulting in delayed diagnosis and enhanced spread of the agent. Small poultry flocks are increasingly common in rural and suburban environments. We assessed the frequency of A. paragallinarum using real-time PCR and clinical conditions present in samples from such flocks submitted to the California Animal Health and Food Safety Laboratory System (Davis, CA) in 2018. From the 294 samples collected for our study, 86 (30%) were PCR-positive for A. paragallinarum. Juvenile birds (≤1 y) were significantly more likely to be PCR-positive ( p = 0.017), and birds diagnosed with respiratory disease had lower Ct values ( p = 0.001) than those without. Concurrent infections were also identified, including with Mycoplasma gallisepticum (18.6%), M. synoviae (18.6%), infectious bronchitis virus (12.8%), and infectious laryngotracheitis virus (7.0%). Only 46.5% of PCR-positive chickens had antemortem respiratory signs, making endemic infections in these flocks highly likely. Our study demonstrates that A. paragallinarum is present in small-flock operations including those without respiratory disease and may present a risk for airborne pathogen transmission to commercial poultry operations.

Download Full-text

Detection and Confirmation of Clostridium botulinum in Water Used for Cooling at a Plant Producing Low-Acid Canned Foods

Applied and Environmental Microbiology ◽

10.1128/aem.00820-10 ◽

2010 ◽

Vol 76 (22) ◽

pp. 7653-7657 ◽

Cited By ~ 10

Author(s):

Amita Sachdeva ◽

Stephanie L. H. Defibaugh-Chávez ◽

James B. Day ◽

Donald Zink ◽

Shashi K. Sharma

Keyword(s):

Real Time ◽

Drug Administration ◽

Water Samples ◽

Real Time Pcr ◽

Clostridium Botulinum ◽

Enzyme Linked Immunosorbent Assay ◽

Mouse Bioassay ◽

Screening Methods ◽

Canned Foods ◽

The U.S

ABSTRACT Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.

Download Full-text

Real-Time and Conventional PCR Tools for Detection and Discrimination of Calonectria pseudonaviculata and C. henricotiae Causing Boxwood Blight

Plant Disease ◽

10.1094/pdis-09-19-2053-re ◽

2021 ◽

Vol 105 (1) ◽

pp. 164-168 ◽

Cited By ~ 1

Author(s):

Yonghong Guo ◽

Margaret Pooler

Keyword(s):

Economic Impact ◽

Real Time ◽

Real Time Pcr ◽

Internal Transcribed Spacer ◽

Histone H3 ◽

Fungal Species ◽

Species Specificity ◽

Conventional Pcr ◽

The U.S ◽

Β Tubulin

Calonectria pseudonaviculata and C. henricotiae are the causal agents of boxwood blight, a devastating disease of boxwood that has caused significant economic impact on the nursery and landscape industries in the U.S. and in Europe. The two species are genetically distinct and are found in different geographic areas but are difficult to distinguish based on morphology and pathogenicity. Fast, accurate, and inexpensive methods to detect and differentiate these species is critical in stopping the spread of the disease. We designed primer pairs based on available sequences of four conserved regions—calmodulin, histone H3, internal transcribed spacer, and β-tubulin—and tested their ability to differentiate the two Calonectria species. Here we report three primer pairs derived from sequence differences in the histone H3 region that can be used to specifically detect C. pseudonaviculata, C. henricotiae, or both species. Specificity of these primers was tested against nine isolates of C. pseudonaviculata, three isolates of C. henricotiae, 13 other Calonectria species, and five isolates from related genera using conventional and real-time PCR. These are the first primers available that can be used with either a multiplexed conventional PCR or SYBR-based real-time PCR to specifically detect and differentiate the two fungal species.

Download Full-text

Detection of Bordetella avium by TaqMan real-time PCR in tracheal swabs from wildlife birds

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2017-0005 ◽

2017 ◽

Vol 20 (1) ◽

pp. 31-36 ◽

Cited By ~ 2

Author(s):

T. Stenzel ◽

D. Pestka ◽

B. Tykałowski ◽

M. Śmiałek ◽

A. Koncicki ◽

...

Keyword(s):

Respiratory Tract ◽

Real Time ◽

Real Time Pcr ◽

Genetic Material ◽

Positive Control ◽

Farm Animals ◽

Diagnostic Laboratory ◽

Bacterial Dna ◽

Bordetella Avium ◽

Low Prevalence

Abstract Bordetella avium, the causing agent of bordetellosis, a highly contagious infection of the respiratory tract in young poultry, causes significant losses in poultry farming throughout the world. Wildlife birds can be a reservoir of various pathogens that infect farm animals. For this reason the studies were conducted to estimate the prevalence of Bordetella avium in wildlife birds in Poland. Tracheal swab samples were collected from 650 birds representing 27 species. The bacterial DNA was isolated directly from the swabs and screened for Bordetella avium by TaqMan real-time PCR. The assay specificity was evaluated by testing DNA isolated from 8 other bacteria that can be present in avian respiratory tract, and there was no amplification from non-Bordetella avium agents. Test sensitivity was determined by preparing standard tenfold serial dilutions of DNA isolated from positive control. The assay revealed to be sensitive, with detection limit of approximately 4.07x10^2 copies of Bordetella avium DNA. The genetic material of Bordetella avium was found in 54.54% of common pheasants, in 9.09% of Eurasian coots, in 3.22% of black-headed gulls and in 2.77% of mallard ducks. The results of this study point to low prevalence of Bordetella avium infections in wildlife birds. The results also show that described molecular assay proved to be suitable for the rapid diagnosis of bordetellosis in the routine diagnostic laboratory.

Download Full-text