Development, optimization, and validation of a <i>Classical swine fever virus</i> real-time reverse transcription polymerase chain reaction assay

Eberling, August J.; Bieker-Stefanelli, Jill; Reising, Monica M.; Siev, David; Martin, Bruce L.; McIntosh, Michael T.; Beckham, Tammy R.

doi:10.1177/1040638711416970

Cited by 7 publications

(11 citation statements)

References 10 publications

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“…The mRT-qPCR consisted of previously well-characterized and published assays 1,4,21,31 that, at the time of this publication, were in use as single-plex RT-qPCR or qPCR at the USDA-NVSL-FADDL and within the U.S. National Animal Health Laboratory Network (NAHLN). These assays were combined into the multiplex format (Table 2), and the linear dynamic range and relative analytical sensitivity of the mRT-qPCR assay was determined using serial dilutions of pooled purified nucleic acid (purNA) from culture stock viruses.…”

Section: Multiplex Rt-qpcr Linear Dynamic Range Relative Analytical mentioning

confidence: 99%

“…Primer and TaqMan probe sequences for detection of ASFV, CSFV, FMDV, and XIPC were taken from previous publications, 1,4,20,21 and optimal oligonucleotide concentrations were determined through empirical testing. Fluorescence and/or quencher molecules for oligonucleotide probes for ASFV and FMDV were modified from their original USDA designs in order to make the mRTqPCR compatible for simultaneous discrimination of 4 different targets.…”

Section: Oligonucleotidesmentioning

confidence: 99%

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Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

et al. 2015

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Abstract. African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations.

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Section: Multiplex Rt-qpcr Linear Dynamic Range Relative Analytical mentioning

confidence: 99%

Section: Oligonucleotidesmentioning

confidence: 99%

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

et al. 2015

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“…Grau et al developed a multiplex real-time PCR (mRT-qPCR) for concurrent detection of ASFV, classical swine fever virus, and foot and mouth disease virus, and evaluated use of this assay on swine oral secretions (116). This mRT-qPCR consisted of previously published singleplex RT-qPCR or qPCR assays (117)(118)(119) in use at the USDA-NVSL-FADDL and within the NAHLN, that for this study were combined into the multiplex format (116). A multi-plexed real time PCR assay has also been developed for ASFV and classical swine fever virus (120).…”

Section: Molecular Detectionmentioning

confidence: 99%

Rift Valley Fever Virus, Japanese Encephalitis Virus, and African Swine Fever Virus: Three Transboundary, Vector-Borne, Veterinary Biothreats With Diverse Surveillance, and Response Capacity Needs

2019

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Early detection of emerging foreign animal diseases is critical to pathogen surveillance and control programs. Rift valley fever virus (RVFV), Japanese encephalitis virus (JEV), and African swine fever virus (ASFV) represent three taxonomically and ecologically diverse vector-borne viruses with the potential to be introduced to the United States. To promote preparedness for such an event, we reviewed the current surveillance strategies and diagnostic tools in practice around the world for these emerging viruses, and summarized key points pertaining to the availability of existing guidelines and strategic approaches for early detection, surveillance, and disease management activities. We compare and contrast the surveillance and management approaches of these three diverse agents of disease as case studies to emphasize the importance of the ecological context and biology of vectors and vertebrate hosts. The information presented in this review will inform stakeholders of the current state of surveillance approaches against these transboundary foreign animal disease which threaten the United States.

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“…Different Taqman real time RT-PCR assays have been reported to detect C-strain vaccines or/and wild-type viruses (Cheng et al, 2008;Depner et al, 2007;Eberling et al, 2011;Hoffmann et al, 2011;Pérez et al, 2011). The Taqman probes were designed based on the mutations of single or multiple positions of the target nucleotide sequences and can detect single nucleotide polymorphism (SNP).…”

Section: Discussionmentioning

confidence: 99%

Development and Application of a RT-NestPCR Assay for Differential Detection of C-Strain and Wild-Type Viruses of Classical Swine Fever Virus

Wang¹,

Yi-bao²,

Cong³

et al. 2013

SAR

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Due to the urgent need of differentiation of infected from vaccinated animals in control and eradication of classical swine fever (CSF) and the shortcomings of current differential diagnostic tools, this study is aiming to establish a RT-nestPCR assay for differential detection of wild-type viruses and lapinized Chinese vaccine strain (C-strain) of classical swine fever virus (CSFV) of high sensitivity. Two pairs of CSFV-specific primers were designed in the conservative regions of NS5B (a non-structural protein encoded by the CSFV genome, which performs the RNA dependent RNA polymerase activity) and 3′ un-translated regions (3′-UTR) to encompass the T-rich insertion uniquely existing in the 3′-UTR of C-strain genome. Thus the amplification fragment of C-strain is longer than that of the wild-type viruses for it contains the T-rich insertion region. Two pairs of primers were used in combination and the wild-type viruses and C-strain of CSFV could be detected and accurately distinguished with a high sensitivity through super fine resolution (SFR) argarose gel electrophoresis that displays the different lengths of the amplicons. The detection limit of the C-strain and Shimen strain were respectively 4.5×10 -2 pg and 3.2×10 -2 pg of viral RNA. The results of the specificity test showed that this method can detect different strains of CSFV without amplifying other non-CSFV pathogens. The results of the detection of 400 clinical samples indicated that 16 samples were CSFV positive in total; in which 4 samples were C-Strain positive and 12 were wild-type CSFV positive. The total CSFV positive rate was 4%. The detection results of the 14 batches of C-Strain vaccines showed that all samples displayed bands of C-Strain amplicons in the SFR argarose gel electrophoresis and all vaccines were free of wild-type virus contamination. In conclusion, the RT-nestPCR assay established in the present study could supply a sensitive and specific test method for distinguishing wild-type CSFV infected animals from those vaccinated with C-strain vaccines in the field.

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Development, optimization, and validation of a Classical swine fever virus real-time reverse transcription polymerase chain reaction assay

Cited by 7 publications

References 10 publications

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

Rift Valley Fever Virus, Japanese Encephalitis Virus, and African Swine Fever Virus: Three Transboundary, Vector-Borne, Veterinary Biothreats With Diverse Surveillance, and Response Capacity Needs

Development and Application of a RT-NestPCR Assay for Differential Detection of C-Strain and Wild-Type Viruses of Classical Swine Fever Virus

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