Reverse transcriptase-PCR assay for detection of hog cholera virus

Harding, Mark J.; Lutze-Wallace, Cyril; Prud'homme, Isabelle; Zhong, Xiaohui; Rola, J.

doi:10.1128/jcm.32.10.2600-2602.1994

Cited by 31 publications

(6 citation statements)

References 16 publications

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“…Previous work on the RT-PCR of CSFV developed in the laboratory of the authors (5,6) indicated that the test is highly specific. Testing was conducted for viruses other than CSFV and all samples gave negative results.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus

Vydelingum¹,

Tang²,

K³

et al. 1998

Rev. Sci. Tech. OIE

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The authors evaluated the ability of a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect classical swine fever virus (CSFV) in comparison with virus isolation and detection by an indirect immunoperoxidase assay (VI-IPA).To determine the specificity of the assay, samples from 60 spleens, 45 tonsils, ten submandibular lymph nodes, eight mesenteric lymph nodes and four kidneys, collected from pigs of various ages which had been slaughtered in abattoirs in Canada (a population free from CSFV), were tested. All the samples tested gave negative results by both VI-IPA and RT-PCR. A total of 20 samples were passaged in porcine kidney (PK) 15 cells and retested by both assays. All were found to be negative, giving a specificity of 100%. To determine the analytical sensitivity of the assay, a similar comparative study was conducted, using CSFV grown in tissue culture and tonsil tissues from a CSFV-infected pig. For both infected tissues and tissue culture fluids, RT-PCR was ten times more sensitive than VI-IPA. Amounts as small as 0.6 infectious units per 100 mg of tissue were detected by RT-PCR, compared to 6 infectious units by VI-IPA. Similarly, RT-PCR could detect as little as 0.1 infectious unit per ml in tissue culture fluids, compared to one infectious unit per ml by VI-IPA. To determine diagnostic sensitivity, three coded panels (two internal and one external), comprising 45 samples from 14 pigs, were tested. The diagnostic sensitivity of both RT-PCR and VI-IPA was found to be 100% for both internal panels. The results of the external panel, apart from two samples that were missed by both RT-PCR and VI-IPA, were found to be in total agreement. These two samples remained negative after amplification in PK15 cells. All the RT-PCR results were based on a single test whereas, for the VI-IPA results, positive results were obtained for five samples only after an amplification round in PK15 cells. Application of the RT-PCR assay for the diagnosis of CSFV would enable improved detection of the virus in a shorter time period.

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Section: Discussionmentioning

confidence: 99%

“…The RT-PCR assay for the detection of CSFV has been previously described (5). In brief, the PCR primers are derived from the pl25 gene of the alfort and Brescia strains (9,11), i.e.…”

Section: Reverse Transcription-polymerase Chain Reaction and Analysismentioning

confidence: 99%

Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus

Vydelingum¹,

Tang²,

K³

et al. 1998

Rev. Sci. Tech. OIE

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“…Rapid and pre-clinical laboratory diagnosis of CSFV is therefore a matter of urgency in order to prevent and control the epidemics. Current methods for diagnosis of CSFV rely on virus isolation [12], fluorescent antibody technique [13], enzyme-linked immunosorbent assay [14,15], RT-PCR [7,[16][17][18][19][20] and real-time RT-PCR [21]. RT-PCR and real-time RT-PCR procedures are generally considered to be the most sensitive in vitro method for detecting CSFV infection.…”

Section: Introductionmentioning

confidence: 99%

Rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification

Chen¹,

Zhang²,

Ma³

et al. 2009

Molecular and Cellular Probes

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The usefulness of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid pre-clinical detection of classical swine fever virus (CSFV) infection was evaluated. The RT-LAMP reaction could be finished in 60 min under isothermal condition at 65 degrees C by employing a set of four primers targeting the 5' untranslated region of CSFV. The RT-LAMP assay of CSFV showed higher sensitivities than that of RT-PCR, with a detection limit of 5 copies per reaction. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 2, porcine parvovirus, porcine pseudorabies virus, Japanese encephalitis virus, and porcine reproductive and respiratory syndrome virus. The detection rates of CSFV RT-LAMP, RT-PCR and virus isolation for samples including blood, tonsil, nasal and rectal swabs from uninoculated pigs without any clear clinical symptom were 89%, 78% and 71%, respectively. Furthermore, all of the assays showed higher sensitivity for blood and tonsil swabs samples than nasal and rectal swabs. These results indicate that the CSFV RT-LAMP assay is a valuable tool for its rapid, cost-effective detection and has potential usefulness for rapid pre-clinical detection and surveillance of classical swine fever in developing countries.

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“…9 Some of the current diagnostic methods for CSF include the detection of viral antigens in tonsils using fluorescent conjugated antibody, antigen capture enzyme-linked immunosorbent assays (ELISAs), 2 or detection of genomic RNA by reverse transcription polymerase chain reaction (RT-PCR). 5,6,10 The disease may present in a peracute, acute, subacute, chronic, or persistent form. Subacute and chronic forms of CSF are often associated with previously vaccinated herds or low virulence viruses and may remain clinically undetected.…”

mentioning

confidence: 99%

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

et al. 2011

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Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a well-characterized assay that can deliver a rapid, accurate diagnosis prior to the onset of clinical signs. A real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription polymerase chain reaction (RT-PCR) for CSF was developed by the United States Department of Agriculture (USDA) at the Plum Island Animal Disease Center (CSF PIADC assay) and evaluated for analytical and diagnostic sensitivity and specificity. A well-characterized panel including Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) isolates was utilized in initial feasibility and optimization studies. The assay was initially designed and validated for use on the ABI 7900HT using the Qiagen QuantiTect® Probe RT-PCR chemistry. However, demonstrating equivalency with multiple one-step RT-PCR chemistries and PCR platforms increased the versatility of the assay. Limit of detection experiments indicated that the Qiagen QuantiTect® Multiplex (NoROX) and the Invitrogen SuperScript® III RT-PCR kits were consistently the most sensitive one-step chemistries for use with the CSF PIADC primer/probe set. Analytical sensitivity of the CSF PIADC assay ranged from <1–2.95 log10 TCID50/ml on both the ABI 7900HT and ABI 7500 platforms. The CSF PIADC assay had 100% diagnostic sensitivity and specificity when tested on a panel of 152 clinical samples from the Dominican Republic and Colombia. The ability to perform this newly developed assay in 96-well formats provides an increased level of versatility for use in CSF surveillance programs.

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Reverse transcriptase-PCR assay for detection of hog cholera virus

Cited by 31 publications

References 16 publications

Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus

Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus

Rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification

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

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