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An updated TaqMan real-time PCR for canine and feline parvoviruses.

ABSTRACT: Canine parvovirus type 2 (CPV-2) emerged in late 1970s from the feline panleukopenia virus (FPLV) and developed, since then, into novel genetic and antigenic variants (CPV-2a, -2b and -2c). Canine and feline parvoviruses cause an acute enteric disease in their hosts, with high level of viral shedding. In this study, a quantitative TaqMan PCR for detection and quantitation of canine and feline parvoviruses in serum and fecal samples was developed. The primers were designed based upon the entire GenBank content for CPV and FPLV. A standard curve was generated, and validation tests were performed using 10-fold serial dilutions of CPV-2 virus in CPV/FPLV-negative feces and CPV/FPLV-negative serum samples. As a result, the 100% detection limit of the PCR was 18 copies of the viral genome per ?l of serum and fecal sample. All canine parvovirus types as well as FPLV were detected. In conclusion, the real-time PCR represents an upgraded and useful tool to identify and quantify canine and feline parvoviruses in different sample matrices.

SUBMITTER: Streck AF

PROVIDER: S-EPMC7119788 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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An updated TaqMan real-time PCR for canine and feline parvoviruses.

Streck André Felipe AF Streck André Felipe AF Rüster Dana D Truyen Uwe U Homeier Timo T

Journal of virological methods 20130513 1

Canine parvovirus type 2 (CPV-2) emerged in late 1970s from the feline panleukopenia virus (FPLV) and developed, since then, into novel genetic and antigenic variants (CPV-2a, -2b and -2c). Canine and feline parvoviruses cause an acute enteric disease in their hosts, with high level of viral shedding. In this study, a quantitative TaqMan PCR for detection and quantitation of canine and feline parvoviruses in serum and fecal samples was developed. The primers were designed based upon the entire G ...[more]

PMID: 23680092

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Project description:BackgroundGene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.ResultsRNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), beta-actin (ACTB), beta-2-microglobulin (B2M), beta-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.ConclusionThe reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.

Project description:BackgroundMorbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.ResultsA specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.ConclusionsThe TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

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An updated TaqMan real-time PCR for canine and feline parvoviruses.

Publications

An updated TaqMan real-time PCR for canine and feline parvoviruses.

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