Rapid Detection of Classical Swine Fever Virus by a Portable Real-Time Reverse Transcriptase PCR Assay

Risatti, Guillermo R.; Callahan, Johnny D.; Nelson, William M.; Borca, Manuel V.

doi:10.1128/jcm.41.1.500-505.2003

Cited by 71 publications

(66 citation statements)

References 30 publications

(25 reference statements)

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“…In the last decade, various PCR assays have been developed for the detection of ASFV [1,18,35,37] and of CSFV [16,17,19,23,30,38]. These assays provide novel diagnostic tools, but a disadvantage is the restricted ability to detect only a single virus, ASFV or CSFV.…”

Section: Discussionmentioning

confidence: 99%

“…The PCR is an alternative rapid detection method of the viruses [6,10]. It has at least the same sensitivity than standard cell cultures and offers much higher sensitivity and specificity than other available rapid methods such as ELISA and immunofluorescence based assays [23,30,35]. Several uniplex PCR-based methods have been reported for ASFV or CSFV detection.…”

Section: Introductionmentioning

confidence: 99%

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A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

Agüero

Fernández²,

Romero

et al. 2004

Vet. Res.

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-The development and standardisation of a novel, highly sensitive and specific one-step hot start multiplex RT-PCR assay is presented for the simultaneous and differential diagnosis of African swine fever (ASF) and Classical swine fever (CSF). The method uses two primer sets, each one specific for the corresponding virus, amplifying DNA fragments different in length, allowing a gel-based differential detection of the PCR products. Universal detection of ASF and CSF virus strains was achieved through selection of primers in conserved viral genome regions. The detection range was confirmed by analysis of a large collection of isolates of the two viruses. The high specificity of the assay was proven by testing related viruses, uninfected cell line cultures and healthy pig tissues. Additional confirmatory tests of the ASF and CSF virus amplicon specificity, based on restriction endonuclease analysis with BsmA I or Ban II, respectively, are also described. The analysis of whole blood and serum samples from experimentally infected animals proved the usefulness of the method for an early diagnosis of both diseases, even before the appearance of the first clinical signs. A study of 150 positive field samples from several ASF and CSF outbreaks showed the suitability of this method for a rapid (less than five hours), sensitive and specific differential diagnosis in clinical samples. In addition, a highly sensitive and specific uniplex RT-PCR for CSFV was also developed and standardised as a powerful tool for fast and early diagnosis of the disease.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

Agüero

Fernández²,

Romero

et al. 2004

Vet. Res.

View full text Add to dashboard Cite

show abstract

“…Rapid, accurate, and pre-clinical laboratory diagnosis of CSFV is therefore a matter of urgency in order to prevent and control the epidemics. Current laboratory diagnoses of CSFV rely on virus isolation, serological methods, detection of antigen and nucleic acid amplification [9][10][11][12]. The PCRbase procedures are generally considered to be the most sensitive in vitro method for detecting CSFV infection.…”

Section: Introductionmentioning

confidence: 99%

Detection of nucleic acid of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Wongsawat¹,

Dharaku²,

Narat³

et al. 2011

Health

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Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5'UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PR-RSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.

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“…SK6 monolayers were infected, overlaid with 0.5% agarose, and incubated at 37°C for 3 days. Plates were fixed with 50% (vol/vol) ethanolacetone and stained by immunohistochemistry with MAb WH303 (30).…”

mentioning

confidence: 99%

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

Risatti

Holinka

Sainz

et al. 2007

J Virol

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E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.

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Rapid Detection of Classical Swine Fever Virus by a Portable Real-Time Reverse Transcriptase PCR Assay

Cited by 71 publications

References 30 publications

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

Detection of nucleic acid of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

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