Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens

Gamble, David A.; Lobbiani, A.; Gramegna, M.; Moore, Lisa E.; Colucci, Giuseppe

doi:10.1128/jcm.35.3.673-675.1997

Cited by 42 publications

(34 citation statements)

References 11 publications

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“…Furthermore, such steps increase the risk of inaccuracy and contamination. Several RT-PCR assays have been developed to detect FCoV (Li and Scott, 1994;Herrewegh et al, 1995;Gamble et al, 1997). Two of those assays are nested PCRs and in all of those ethidium bromide staining and UV light transillumination of electrophoretically separated PCR products is necessary to detect PCR products.…”

Section: Discussionmentioning

confidence: 99%

“…Several polymerase chain reaction (PCR) based methods have been suggested in detection of the positive-stranded FCoV RNA, but none of these were designed to be quantitative (Li and Scott, 1994;Herrewegh et al, 1995;Gamble et al, 1997). In addition, conventional PCR methods are time consuming due to several post-amplification steps, contain a certain risk of cross-contamination between the samples due to a separate labor-intensive reverse transcription (RT) step and a second PCR step in nested PCR systems, are limited in sensitivity and allow only relatively few samples to be processed at one time.…”

Section: Introductionmentioning

confidence: 99%

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One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses

Gut

Leutenegger

Huder

et al. 1999

Journal of Virological Methods

160

154

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A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Forster-type energy transfer. During the polymerase extension steps the Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (C(T) value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism 7700 Sequence Detector. After completion of amplification, the C(T) values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses

Gut

Leutenegger

Huder

et al. 1999

Journal of Virological Methods

160

154

View full text Add to dashboard Cite

show abstract

“…Our PCR technique using primers targeted to conserved regions of the viral genome, the 3 -UTR (Lai and Cavanagh, 1997), and its modifications (using the S gene (Gamble et al, 1997)) became a valuable tool for the detection of FCoV in body fluids and tissue samples. Unfortunately, the technique detects also avirulent FCoVs in healthy cats.…”

Section: Introductionmentioning

confidence: 99%

A mRNA PCR for the diagnosis of feline infectious peritonitis

Simons

Vennema

Rofina

et al. 2005

Journal of Virological Methods

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A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The assay is evaluated as a diagnostic test for feline infectious peritonitis (FIP). It is based on a well-documented key event in the development of FIP: the replication of virulent FCoV mutants in monocytes/macrophages. To detect most feline coronavirus field strains, the test was designed to amplify subgenomic mRNA of the highly conserved M gene. The test was applied to 1075 feline blood samples (424 from healthy, 651 from sick cats suspected of FIP) and returned 46% of the diseased cats as positive for feline coronavirus mRNA in their peripheral blood cells; of the healthy cats, 5% tested positive. Of a group of 81 animals in which FIP had been confirmed by post-mortem examination, 75 (93%) tested positive, whereas 17 cats with different pathologies (non-FIP cases) all tested negative. In view of the low rate of false-positive results (high specificity) the mRNA RT-PCR may be a valuable addition to the diagnostic arsenal for FIP.

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“…The recently developed reverse transcriptase polymerase chain reaction (RT-PCR) assays, using primers targeted to highly conserved regions of the viral genome (3 -UTR (untranslated region) (Herrewegh et al, 1995;Fehr et al, 1996), or S-protein gene (Li and Scott, 1994;Gamble et al, 1997)), which are common to all FCoV strains, became a valuable tool for the detection of FCoV nucleic acid in blood, body cavity effusions, faeces and tissue samples of infected cats.…”

Section: Introductionmentioning

confidence: 99%

Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis

Benetka

Kübber‐Heiss

Kolodziejek

et al. 2004

Veterinary Microbiology

110

106

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Feline coronaviruses (FCoV) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (FIP). Independent of virulence variations they are separated into two different types, type I, the original FCoV, and type II, which is closely related to canine coronavirus (CCV). The prevalence of FCoV types in Austrian cat populations without FIP has been surveyed recently indicating that type I infections predominate. The distribution of FCoV types in cats, which had succumbed to FIP, however, was fairly unknown. PCR assays have been developed amplifying parts of the spike protein gene. Type-specific primer pairs were designed, generating PCR products of different sizes. A total of 94 organ pools of cats with histopathologically verified FIP was tested. A clear differentiation was achieved in 74 cats, 86% of them were type I positive, 7% type II positive, and 7% were positive for both types. These findings demonstrate that in FIP cases FCoV type I predominates, too, nonetheless, in 14% of the cases FCoV type II was detected, suggesting its causative involvement in cases of FIP.

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Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens

Cited by 42 publications

References 11 publications

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses

A mRNA PCR for the diagnosis of feline infectious peritonitis

Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis

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