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, Montserrat; Fernández, Jovita; Romero, Luis; Zamora, Mª Jesús; Sánchez, Carmen; Belák, Sándór; Arias, Marisa; Sánchez-Vizcaíno, José Manuel

doi:10.1051/vetres:2004031

Cited by 63 publications

(33 citation statements)

References 32 publications

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“…Due to the fact that China is free from the virus, the related research is limited. The primers of the experiment were selected from the conserved sequence based on foreign related studies (Aguero et al 2004). While detecting PRV, CSFV, PRSSV, PPV, PCV2 and JEV, ASFV may be monitored for.…”

Section: Discussionmentioning

confidence: 99%

A multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, highly pathogenic porcine reproductive and respiratory syndrome virus, porcine reproductive and respiratory syndrome virus and pseudorabies in swines

Hu¹,

Lin²,

Yang³

et al. 2015

Polish Journal of Veterinary Sciences

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In this assay, we developed and evaluated a multiplex PCR (mPCR) for its ability in detecting multiple infections of swine simultaneously. Four pairs of primers were used to detect five viruses. Specific primers were designed for classical swine fever virus (CSFV), African swine fever virus (ASFV) and pseudorabies (PRV). A pair of primers was designed prudently for two different types of porcine reproductive and respiratory syndrome virus that respectively were porcine reproductive and respiratory syndrome virus (PRRSV), highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). The detection limits of the mPCR were 1. A total of 49 clinical specimens were tested by the mPCR, and the result showed that co-infection by two or three viruses was 51%. In conclusion, the PCR is a useful tool for clinical diagnosis of not only single infections but also mixed infections in swines.

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Section: Discussionmentioning

confidence: 99%

A multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, highly pathogenic porcine reproductive and respiratory syndrome virus, porcine reproductive and respiratory syndrome virus and pseudorabies in swines

Hu¹,

Lin²,

Yang³

et al. 2015

Polish Journal of Veterinary Sciences

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“…By using multiplex PCR systems, several infectious agents can be detected and differentiated simultaneously in a single reaction, reducing costs and efforts as well as the amount of sample material and time required (37,38). A variety of conventional multiplex PCR assays for the detection and differentiation of different swine viruses based on amplicon size have been developed during recent years (39)(40)(41)(42)(43)(44)(45)(46). However, qPCR assays have several advantages compared to conventional PCR, combining a reduced risk of cross-contamination with a high sensitivity and the possibility of semiquantitative analysis.…”

Section: Discussionmentioning

confidence: 99%

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

Wernike

Hoffmann

2013

J Clin Microbiol

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Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV-and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance.

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“…Detection of amplicons is possible by gel electrophoresis, but nowadays mainly real-time RT-PCR"s are being used. These PCR"s use either SYBR green to detect amplicons, or, for enhanced specificity, hydrolysis of hybridization probes (Liu et al, 1991;Roehe and Woodward, 1991;Katz et al, 1993;Diaz et al, 1998;McGoldrick et al, 1998;Aguero et al, 2004;Belak, 2005).…”

Section: Rt-pcrmentioning

confidence: 99%

Control and eradication of Classic Swine Fever in wild boar

2009

EFSA Journal

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SummaryClassical swine fever (CSF) is a disease that has been causing major socio-economic damages in the EU during the last decades. Although considerable progress has been made in the eradication and prevention of the disease, the threat for an epidemic still exists.The virus is endemic in the wild boar population of several member states (MS). Wild boar cannot be managed as domestic pigs. Hunting and vaccination have been tentatively used in order to stop transmission by reducing the number of susceptibles. Oral vaccination of wild boar with modified live vaccine based on the C-strain (the only suitable) is used; this vaccine does not allow serological differentiation between vaccinated and infected animals.The aim of the control measures for CSF in wild boar is to reduce the risk of transmission to domestic pigs, to prevent an "endemic phase evolution" or to reduce the endemic phase duration.In order to support and to improve the control and eradication measures as regards CSF in wild boar, EFSA was requested by the Commission to provide scientific advice on the efficacy of the available surveillance, hunting and vaccination measures to control and eradicate CSF in feral pig populations (wild boar), considering the possible use of new diagnostic tests and vaccines.In order to reply to the mandate data were collected from MS through two questionnaires (CSF vaccines, hunting practices and CSF vaccination in wild boar). Data were also extracted from the EU CSF wild boar data base, after requesting permission from the participating MS.Considering that the data received were rather limited and not comparable, the WG decided to use a model in order to fill the gaps in the available knowledge. For the evaluation of the efficacy of CSF control in wild boar a continuous metapopulation compartmental model developed in an EU research project (FP6-5015599-CSFVACCINE&WILDBOAR) was applied.Although the disease manifests in the same manner in both domestic pig and wild boar it is more difficult to identify in wild boar because clinical monitoring of these is hardly possible. Wild boar found dead constitutes the main alert sign of CSF. Hunters and gamekeepers should be instructed to report the finding of each dead wild boar to the competent authority at all times. In case of high-risk situations, a passive surveillance should be complemented by an active serological surveillance. The active sampling of wild boar is not as efficient as in domestic pigs considering that hunting is the sole practical system to obtain samples and that the aim of hunting is quite different.Consequently, the sample size is not controlled by authorities and fits rarely the aim of the survey in terms of detecting the presence of disease at a certain level. In addition, the interpretation of serological results is confounded by maternal derived antibodies (until the 6th month of age), vaccination and the sampling quality.There is no definition at EU level of spatial and temporal units for surveillance, neither for criteria defining a disease free wi...

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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

Cited by 63 publications

References 32 publications

A multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, highly pathogenic porcine reproductive and respiratory syndrome virus, porcine reproductive and respiratory syndrome virus and pseudorabies in swines

A multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, highly pathogenic porcine reproductive and respiratory syndrome virus, porcine reproductive and respiratory syndrome virus and pseudorabies in swines

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

Control and eradication of Classic Swine Fever in wild boar

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