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Development of updated porcine reproductive and respiratory syndrome virus products
PRRS is a viral disease that leads to abortions and weak-born piglets, increased mortality in suckling and weaned piglets, and respiratory disease in weaners and finishers. PRRSV belongs to the Arteriviridae family within the order Nidovirales and contains a single-stranded, positive-sense RNA genome of approximately 15 kilobases. PRRSV is split into two genotypes, PRRSV-1 (Type 1) and PRRSV-2 (Type 2), which only share approximately 50% sequence identity and are highly variable within each type. The PRRSV genome encodes ten open reading frames (ORFs). ORF1a and ORF1b encode nonstructural polyproteins with replicase and polymerase activity. ORFs 2, 3, and 4 encode structural glycoproteins GP2, GP3, and GP4, respectively, which are responsible for the formation of a trimeric complex that for viral entry. ORFs 5, 6, and 7 encode the major structural proteins GP5, matrix (M), and nucleocapsid (N), respectively.
qPCR eliminates the need for culture Improved detection of T. foetus by RT-medium
Tritrichomonas foetus (T. foetus) is a protozoan that is the causative agent of bovine trichomoniasis, a sexually transmitted disease found worldwide in bulls and cows. The previous DNA-based Trich detection kit, a quantitative PCR (qPCR) assay, has been considered the gold standard for PCR-based detection of T. foetus in preputial samples (smegma) collected in culture medium. However, in 2018, the Texas Veterinary Medical Diagnostic Laboratory (TVMDL) published primers and probe sequences that target the 5.8S ribosomal RNA rather than the gene, which generates earlier Ct values due to higher concentration of target template when compared to the DNA-targeting qPCR test. Since combining the reverse-transcription qPCR (RT-qPCR) primers and probe design with a one-step RT master mix makes the reaction more sensitive, it eliminates the need to collect and incubate smegma in the InPouch™ commercial culture medium and offers a simple PBS sample collection instead. Additionally, due to the higher target template concentration and the reduction in inhibition, it provides the capability to pool several smegma samples for nucleic acid extraction and RTqPCR.
Evaluation of the VetMAX™-GOLD AIV Detection Kit and VetMAX™-GOLD SIV Detection Kit for the detection of avian and swine influenza viruses
Real-time reverse-transcription polymerase chain reaction (RRT-PCR) assays play an important role in the rapid identification of avian and swine influenza A viruses (AIV and SIV, respectively). The aim of this study was to evaluate the Applied Biosystems™ VetMAX™-Gold AIV Detection Kit and Applied Biosystems™ VetMAX™-Gold SIV Detection Kit from Thermo Fisher Scientific (Figure 1) for detection of AIV and SIV in clinical samples, respectively and the performance of the Applied Biosystems™ VetMAX™-Gold SIV Subtyping Kit on selected strains.
Mastitis Diagnosis utilizing new multiplex qPCR kits for simultaneous detection of up to 15 pathogens
Bovine mastitis is the most common and costly disease among dairy cows. When mastitis is identified in a cow’s quarter(s), it is important to identify the pathogen causing the infection because different categories of pathogens require different management strategies. Applied Biosystems™ VetMAX™ MastiType multiplex qPCR kits, together with the Applied Biosystems™ MagMAX™ CORE Nucleic Acid Purification Kit, are designed to provide same-day results even for Mycoplasma spp., enabling farmers and veterinarians to take fast and well informed action. It provides a rapid workflow that allows diagnosis of milk samples for the presence of 19 different types of microorganisms and penicillin-resistance genes. The extracted DNA can then be tested with three different 4-plexed VetMAX™ MastiTypePCR kits, on either the Applied Biosystems™ 7500 or QuantStudio™ 5 series of Real-Time PCR Systems.
Real-Time PCR Data Analysis Tool Utilizing a Novel Cloud-Based Software for Easy Interpretation of Animal Pathogen Detection
Current software versions on Real-Time PCR instruments were not specifically designed for the detection of pathogens, making data analysis and interpretation of multiplexed Animal Health assays difficult for users. Typically, qPCR data is analyzed manually which can be labor intensive and time-consuming. A complete package of data analysis software provides a faster and much more convenient alternative. The Animal Health group under the Applied Biosystems™ brand at Thermo Fisher Scientific now offers VeriVet Software, a user-friendly solution for the analysis of qPCR data for detecting animal pathogens.
The Use of Modules for the Detection of Pathogens in Mastitic Milk and Mycobacterium avium subsp. Paratuberculosis in Bovine Feces with the MagMAX™ CORE Nucleic Acid Purification Kit
In many testing labs, the kit used to isolate nucleic acids from pathogens depends largely on the sample type and pathogen of interest. The need to use multiple extraction kits reduces efficiency and adds complexity to testing. For example, the product used to isolate nucleic acid from Mycobacterium avium subsp. paratuberculosis (MAP) in bovine feces is different than the product used for mastitis testing in milk, which in turn is different than the product required for Bovine Viral Diarrhea Virus (BVDV) from ear notches. The MagMAX™ CORE Nucleic Acid Purification Kit allows for testing numerous sample types with a single kit that uses a core workflow for all sample types.
Evaluation of GenoTubes for transport, storage and extraction of nucleic acids from Porcine Oral Fluids
Porcine Oral fluids are currently transported and stored in a refrigerated environment, which adds to the cost of shipping and also has a significant environmental impact. In this experiment, we attempt to use GenoTubes to transport Oral Fluids from the producer to the diagnostic lab.
AAVLD Mastitis Workshop
The last sample extraction method you may ever need. Flexible solution designed to meet lab’s current and future testing needs. For widest range of animal health sample matrices. Helps make labs more efficient. Magnetic bead-based sample extraction. Automatable on KingFisher magnetic particle processors.
PRRS virus surveillance: Role of virus sequencing and virus detection by PCR
PRRSV is highly mutating, so we consistently monitor PRRSV strains to be sure to offer the most up-to-date PCR solution to enable our customers to work with confidence and detect all strains of concern. The monitoring of circulating European PRRSV strains using sequencing technologies enables the sequencing of RNA directly isolated from field samples. Sequencing approaches offer the possibility to identify new PRRSV strains, increasing the performance of a diagnostic tool for PRRSV detection. The Applied Biosystems VetMAX PRRSV EU & NA assay in development is designed to reinforce the efficacy of PRRSV surveillance programs in the field, with the detection of the four subtypes of the PRRSV European genotype and a diagnostic sensitivity of 99%. Thermo Fisher Scientific offers a range of adapted workflows from sampling, extraction methods to sequencing solutions.
The Applied Biosystems VetMAX PEDV/TGEV/SDCoV kit was developed using various types of environmental and related samples obtained from the field, and found to yield high specificity and sensitivity. To examine its performance relative to competitors’ solutions, we compared it to two comparable kits prominently used in the market. Environmental, oral fluid, and fecal samples infected with PEDV (N=15), TGEV (N=10), and SDCoV(N=14) were obtained from the field, and negative, positive, and no template controls were included in the testing. RT-qPCR for all three manufacturers’ kits were run on the Applied Biosystems 7500 Fast Real-Time PCR System according to the manufacturer’s recommendations. The VetMAX PEDV/TGEV/SDCoV kit consistently showed equivalent or better results to other kits tested, with consistently lower CTs compared to one of the kits and a higher signal versus baseline noise for the other.
Development and validation of new Tuberculosis real-time PCR kit
Bovine tuberculosis (TB) is a chronic disease affecting cattle, as well as wild animals, and is caused primarily by Mycobacterium bovis. Applied Biosystems™ VetMAX™ M. tuberculosis Complex kit (MTBC), a new molecular tool, allows the simultaneous detection of Mycobacteria belonging to Mycobacterium tuberculosis complex (MTBC) and an internal control. To demonstrate the kit’s performances, verification studies were carried out on cattle and a variety of wild animals.
Development and validation of the VetMAX™-Gold MAP Detection Kit
The USDA-licensed VetMAX™-Gold MAP Detection Kit is a real-time PCR assay for the rapid in vitro detection of MAP DNA purified from bovine feces. The assay targets a unique sequence element in the MAP genome to provide highly sensitive and specific results. The purpose of this study is to determine the performance characteristics of the VetMAX™-Gold MAP Detection Kit in detecting MAP DNA from nucleic acid extracted from individual and pooled bovine fecal samples.
Enable the right result the first time with Xeno Internal Positive Control
Animal sample matrices used for diagnostic qPCR testing may contain a variety of PCR inhibitors that can potentially lead to false-negative results. In an effort to provide a solution to help diagnose farm animal health issues more accurately, we’ve developed an internal positive control that can be easily integrated into existing TaqMan™-based workflows to test for the presence of PCR inhibitors, thereby lowering the risk of false-negative results.
Monitoring of European PRRSV strains using sequencing technologies
PRRS is caused by a single stranded positive-sense RNA enveloped virus with a high mutation rate leading to greater heterogeneity of the nucleotide sequence between the individual strains. The high genetic virus diversity increases the risk of reduced sensitivity for real-time RT PCR diagnostic tool. The aim of the present study was to monitor circulating PRRSV strain throughout Europe using capillary ORF7 sequencing and NGS technology.
Prevalence of Tritrichomonas foetus in beef bulls in the State of Chihuahua, Mexico tested with MagMAX™ Sample Preparation System and VetMAX™-Gold Trich Detection Kit
The prevalence of bovine trichomoniasis in Mexico and particularly in the state of Chihuahua is unknown, since no diagnostic testing has been implemented extensively. Therefore, the aim of this study was to estimate the prevalence of trichomoniasis in beef bulls in the state of Chihuahua with a molecular diagnostic test based on real-time PCR: the VetMAX™-Gold Trich Detection Kit with a United States Department of Agriculture (USDA) license.
Development and validation of a complete workflow solution for SIV testing
We have validated an SIV testing workflow consisting of high-throughput nucleic acid purification, SIV detection, and SIV subtyping from porcine nasal swab samples. SIV can be detected using the USDA-licensed VetMAX™-Gold SIV Detection Kit, a single-tube, one-step real-time RT-PCR kit for rapid and accurate screening for influenza A. The assay targets three independent regions of the SIV genome to dramatically limit the number of false-negatives due to mutation of the viral genome.
Earlier and easier diagnostic tools for herd management of PRRSV: Comparison of sampling and prevalence under field conditions
The main goals of several studies were to validate oral fluid against blood/serum and tissue sampling methods and also to establish a recommendation for oral fluid sampling under field conditions for an earlier diagnostic of PRRSV with an easier sampling method. Thermo Fisher Scientific asked several laboratories and research institutes throughout the world to evaluate the real-time PCR tools on over 800 field samples of different genotypes. Field studies in Europe and North America allowed evaluation of the kit’s performance on oral fluid samples.
New insights into oral fluids as a diagnosis procedure to detect and determine the prevalence of PRRSV under field conditions
Based on experimentation with European and North American strains of porcine reproductive and respiratory syndrome virus (PRRSV), the probability of detecting PRRSV in oral fluids is significantly associated with PRRSV prevalence in serum (p<0.0001). If the serum prevalence is higher than 50%, the probability of detecting this virus in oral fluid samples is close to 1. Therefore, oral fluid sampling is a good tool for detecting PRRSV at herd level, but it is not suitable for determining the prevalence of this disease.
Development and validation of a new African swine fever real-time PCR kit
African swine fever virus (ASFV) is a notifiable, highly contagious disease that can cause significant economic losses. As there is still no vaccine or treatment available, monitoring ASFV by means of diagnosis is the only way to control it, and is of utmost importance. A new duplex real-time PCR kit that targets the p72 gene and an internal control has been developed, and its performance for diagnosis of ASFV has been assessed.
Why the standardization of Trich regulations and laboratory testing methods across state lines is important for beef producers
Dr. Kathy Simmons, Chief Veterinarian, National Cattlemen's Beef Association, Washington DC. Harmonized state trich regulations for the interstate movement of cattle would facilitate cattle movement at the speed of commerce. Well-defined, thoughtful and mutually accepted testing procedures for trich between adjoining states could eliminate redundant testing procedures and reduce the danger to animals and handlers from repeated or unnecessary testing.*
How the state of Kansas moved from no Trich regulations to implementing current working Trich regulations and accepted diagnostic testing methods
Dr. Bill Brown, Animal Health Commissioner, Kansas Department of Agriculture, Topeka Kansas. Overview of Kansas' enactment of new, more comprehensive trich regulations for the intrastate change of ownership and interstate movement and diagnostic testing of cattle. Dr. Brown appointed a trich working group comprised of four veterinarians and four beef producers to spearhead the evaluation in improving the management of trich in the state of Kansas.*
Why standardization of both state Trich regulations and laboratory testing procedures benefit veterinarians and their beef producer clients
Dr. Jeremy VanBoening - Republican Valley Medical Center, Alma / Holbrook / Franklin Nebraska. Dr. VanBoening has discovered great variability in recommended sample-handling protocols among state diagnostic labs. Labs varied on whether or not samples needed to be incubated, whether or not to put on ice, how the samples are shipped and the labs’ preferred collection media. Standardizing lab recommendations for sample collection and handling would improve the quality of samples submitted for testing.*
Development of an Influenza A Sequencing Workflow on Ion PGM™ Sequencer for Improved Surveillance
Complete genome sequencing is crucial for ongoing identification, surveillance and characterization of Influenza A. When sequencing viral genomes, background host nucleic acids may be co-processed during library preparation resulting in a sequencing reaction in which a majority of reads are taken up by the host genome. One solution to this problem is to run a pre-amplification
Influenza A virus detection from oral fluid and nasal swabs in IAV
inoculated pigs
The detection of IAV in swine populations using nasal swab specimens is labor intensive and relatively insensitive in non-febrile pigs (1). As an alternative, oral fluid samples have been shown to be an excellent surveillance sample for several swine respiratory viruses (2,3,4). The objective of this study was to compare the rate of detection of IAV by RT-PCR, virus isolation (VI), and the VetScan® (Abaxis Inc.).
Correlation between a commercial real-time PCR assay and HEYM culture for MAP in bovine faecal samples
Disease control programmes for Mycobacterium avium subspecies paratuberculosis (MAP) rely on accurate and sensitive tools for the detection of infected animals. Culture based detection of MAP takes many weeks whereas PCR enables rapid detection. Several commercial and many user designed real-time PCR assays exist for the detection of MAP in bovine faecal samples.
Establishment of Presumptive Diagnostic Cut-Off for Persistently Infected Cattle
Bovine Viral Diarrhea Virus causes infection in cattle that has led to major economic losses in both the beef and dairy industries. In utero BVDV infection can induce immunotolerance, causing animals to be persistently infected (PI) for life. PI animals continuously shed the virus and are the main source of BVDV infection in herds. Animals that are acutely or transiently infected will pass the disease and will show
Pooling of Tritrichomonas foetus Cultured Samples Followed by MagMAX™ Sample Preparation System and Amplification with Applied Biosystems qPCR Reagents
The objectives of this study were 1) to compare multiple sample preparation workflows (boiling, QIAGEN, MagMAX™) and real-time PCR assays currently used by diagnostic testing labs with MagMAX™ and Applied Biosystems VetMAX™reagents for individual T. foetus testing, and (2) to assess the feasibility of.
Standardized Sample Preparation For Multiple Sample Matrices
There are many different methods to process different sample matrices. This can lead to confusion and frustration for researchers working with multiple sample types. This abstract describes the MagMAX™ Pathogen RNA/DNA kit which is designed to achieve a more standardized solution so labs can order just one kit for a variety of sample matrices as well as different input volumes for each sample. This will allow labs to work.
*This presentation does not constitute or imply an endorsement or recommendation of Life Technologies products or services by the author. Life Technologies and its affiliates are not endorsing, recommending, or promoting any use or application of Life Technologies products presented by third parties, and information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.