Real-Time PCR (Polymerase Chain Reaction) is a molecular technique used to amplify and quantify DNA sequences in horses. This method allows for the detection and measurement of specific genetic material in real-time, providing valuable insights into genetic expression, pathogen presence, and disease diagnosis. In equine research, Real-Time PCR is utilized to study various aspects such as infectious diseases, genetic disorders, and gene expression profiles. The technique's sensitivity and specificity enable researchers to accurately assess the genetic material of interest, facilitating advancements in equine health diagnostics and management. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings related to Real-Time PCR in equine science.
Leutenegger CM, von Rechenberg B, Huder JB, Zlinsky K, Mislin C, Akens MK, Auer J, Lutz H.Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and emb...
Pusterla N, Leutenegger CM, Chae JS, Lutz H, Kimsey RB, Dumler JS, Madigan JE.This paper describes the kinetics of the human granulocytic ehrlichiosis agent in the blood of horses experimentally infected by intravenous inoculation with infected leukocytes and by infected ticks as evaluated by using a real-time quantitative PCR assay. The data obtained indicated differences in the period of incubation, duration of rickettsemia, and initial and maximal ehrlichial loads between the two routes of infection.
Starick E.Serum samples from 72 stallions were examined for the occurrence of antibodies against equine arteritis virus, of which 41 animals (57%) were found to be positive. 32 of the seropositive stallions were then screened for persistent EAV infection, before and after the breeding season. Semen samples were investigated by RT-PCR followed by dot blot hybridization and nested PCR, and by virus isolation on cell cultures as well. The carrier state was virologically confirmed in 11 of 32 stallions (34%) during the first and in 9 of 20 (45%) during the second investigation. RT-PCR followed by confirmato...
Giguère S, Prescott JF.A reverse transcription-competitive polymerase chain reaction (RT-cPCR) method was developed to quantitate equine interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p35, IL-12 p40, interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), and beta-actin mRNA expression. Using primers based on equine-specific sequences, these cytokines could be detected in concanavalin A-stimulated peripheral blood mononuclear cells. The specificity of the amplified product was confirmed by sequencing. For each cytokine, the assay was made quantitative by generating competitor ...
Smith TA, Davis E, Carpenter S.Lentiviruses replicate in cells of the immune system, and activation of immune cells has been shown to modulate virus replication. To determine the effects of macrophage activation on replication of equine infectious anaemia virus (EIAV), primary horse macrophage cultures (HMCs) were established from 20 different horses, infected with an avirulent strain of EIAV, and stimulated with 5 microg/ml of bacterial endotoxin. Supernatants collected from HMCs were assayed for the presence of tumour necrosis factor (TNF-alpha) and for production of infectious virus. Results indicated that EIAV replicati...
Yamanouchi K, Yoshida S, Hasegawa T, Ikeda A, Chang KT, Matsuyama S, Nishihara M, Miyazawa K, Takahashi M.cDNA encoding equine inhibin alpha-subunit precursor protein was isolated from an equine ovarian cDNA library. For screening, the DNA probe was amplified by the RT-PCR using primers designed based on the rat inhibin alpha-subunit cDNA sequence. Out of 1.2 x 10(5) plaques screened, 19 positive clones were isolated, and one of these clones (Eq-alpha-11) contained a complete open reading frame encoding 367 amino acids. The similarity of the deduced amino acid sequences of both equine inhibin alpha-subunit precursor protein and the mature protein were greater than 80% to those of other six mammali...
The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen...
Li S, Guo K, Wang X, Lin Y, Wang J, Wang Y, Du C, Hu Z, Wang X.Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based ...
Dorrego A, Camino E, Gago P, Buendia-Andres A, Acurio K, Gonzalez S, de Juan L, Cruz-Lopez F.Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, E...
Lawton K, Runk D, Hankin S, Mendonsa E, Hull D, Barnum S, Pusterla N.The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental...
De Coster T, Van Poucke M, Bogado Pascottini O, Angel-Velez D, Van den Branden E, Peere S, Papas M, Gerits I, Govaere J, Peelman L, Vermeesch JR....In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that c...
Kinoshita Y, Niwa H, Uchida-Fujii E, Ueno T. is the predominant equine sp. isolated from clinically normal horses and is suspected to be associated with inflammatory airway disease in which cough is the primary sign. Quantitative evaluation of bacterial counts is useful in assessing the association between the bacteria in samples and observed clinical signs, but this evaluation has been difficult with conventional culture methods of given the need for pre-enrichment using liquid cultures. We established a quantitative real-time PCR (qPCR) assay for the quantification of , targeting the hypothetical protein FJM08_00025. We confirmed it...
Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared betwee...
Zu H, Sun R, Li J, Guo X, Wang M, Guo W, Wang X. subspecies () is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the gene, for the rapid detection of nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the gene, enhancing the overall specificity and sensitivity of the ...
