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Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus.

ABSTRACT: Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats and is associated with interstitial pneumonia, oral ulceration and polyarthritis. Recently, outbreaks have involved a highly virulent FCV that leads to multisystemic signs. Virus isolation and conventional RT-PCR are the most common methods used for FCV diagnosis. However, real-time RT-PCR offers a rapid, sensitive, specific and easy tool for nucleic acid detection. The objective of this study was to design a TaqMan probe-based, real-time RT-PCR assay for detection of FCV. It was determined in our previous study that the first 120 nucleotides of the 5' region of the genome are highly conserved among FCV isolates. Primers and a probe specific for this region were designed for a real-time RT-PCR assay to detect FCV. Initial validation was done using 15 genetically diverse isolates. Also, 122 samples were tested by the new assay and virus isolation. The real-time RT-PCR assay was as sensitive and specific as virus isolation and was far more rapid. This real-time RT-PCR assay targeting the conserved 5' region of the genome is a fast, economical and accurate method for detection of FCV.

SUBMITTER: Abd-Eldaim MM

PROVIDER: S-EPMC7086925 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus.

Abd-Eldaim Mohamed M MM Wilkes Rebecca P RP Thomas Kathy V KV Kennedy Melissa A MA

Archives of virology 20090301 4

Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats and is associated with interstitial pneumonia, oral ulceration and polyarthritis. Recently, outbreaks have involved a highly virulent FCV that leads to multisystemic signs. Virus isolation and conventional RT-PCR are the most common methods used for FCV diagnosis. However, real-time RT-PCR offers a rapid, sensitive, specific and easy tool for nucleic acid detection. The objective of this study was to design a T ...[more]

PMID: 19253013

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Project description:BACKGROUND: Gene 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. RESULTS: RNA 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. CONCLUSION: The 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:BackgroundEquine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections.ResultsThree rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml).ConclusionThe newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.

Dataset Information

Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus.

Publications

Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus.

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