Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses

Liu, L.; Luo, Yuzi; Accensi, Francesc; Ganges, Llilianne; Rodrı́guez, Fernando; Huang, Shan; Ståhl, Karl; Qiu, Hua‐Ji; Belák, Sándór

doi:10.1111/tbed.12538

Cited by 21 publications

(17 citation statements)

References 11 publications

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“…Recently, field applicable RT-PCRs [184] but also alternatives have been designed such as loop-mediated isothermal amplification (LAMP) assays [185,186,187,188,189,190], primer-probe energy transfer RT-qPCR [191,192] or recently insulated isothermal RT-qPCR [193]. Moreover, CSFV can be isolated on different permanent cell lines such as porcine kidney cell lines PK15 or SK6 (Technical Annex to Commission Decision 2002/106/EC).…”

Section: Diagnosismentioning

confidence: 99%

Classical Swine Fever—An Updated Review

Blome

Staubach

Henke

et al. 2017

Viruses

202

217

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Classical swine fever (CSF) remains one of the most important transboundary viral diseases of swine worldwide. The causative agent is CSF virus, a small, enveloped RNA virus of the genus Pestivirus. Based on partial sequences, three genotypes can be distinguished that do not, however, directly correlate with virulence. Depending on both virus and host factors, a wide range of clinical syndromes can be observed and thus, laboratory confirmation is mandatory. To this means, both direct and indirect methods are utilized with an increasing degree of commercialization. Both infections in domestic pigs and wild boar are of great relevance; and wild boars are a reservoir host transmitting the virus sporadically also to pig farms. Control strategies for epidemic outbreaks in free countries are mainly based on classical intervention measures; i.e., quarantine and strict culling of affected herds. In these countries, vaccination is only an emergency option. However, live vaccines are used for controlling the disease in endemically infected regions in Asia, Eastern Europe, the Americas, and some African countries. Here, we will provide a concise, updated review on virus properties, clinical signs and pathology, epidemiology, pathogenesis and immune responses, diagnosis and vaccination possibilities.

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Section: Diagnosismentioning

confidence: 99%

Classical Swine Fever—An Updated Review

Blome

Staubach

Henke

et al. 2017

Viruses

202

217

View full text Add to dashboard Cite

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“…Concordant results were obtained showing a good consistency between the two nucleic acids extraction methods (Table S1). Previous studies have demonstrated a slightly better performance of the bead-based protocol (Liu et al, 2017). Further application of the protocol to process the 64 blood samples that had been collected during previous outbreak investigations, demonstrated the practicability and ease of performing the protocol at NADDEC.…”

Section: Resultsmentioning

confidence: 95%

Overcoming the challenges of pen‐side molecular diagnosis of African swine fever to support outbreak investigations under field conditions

Atim

LeBlanc

Rauh

et al. 2018

Transbound Emerg Dis

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African swine fever (ASF) is a devastating disease of pigs. Without a vaccine, early detection and rapid diagnosis of ASF is a crucial step towards effective disease control. In many countries where ASF is endemic, laboratory infrastructure including sampling and sample shipment is inadequate, and a rapid laboratory confirmation would require that the diagnosis is performed at regional laboratories close to the pig farms of concern, or even at the farm‐side. This study intended to evaluate measures including sample preparation methods, a dried‐down assay, and a portable, battery‐powered real‐time PCR instrument, to improve molecular diagnosis under field conditions. A simple dilution of blood samples, either in Phosphate‐buffered saline or a commercial buffer, worked similarly to beads‐based nucleic acid extraction using a magnet as the core equipment; the latter method did work as well for those samples with low viral load or high Ct values. The real‐time PCR assay using a Universal ProbeLibrary (UPL) probe tolerated suspected inhibitory substances present in the prepared samples better, whereas the dried‐down assay had a higher diagnostic sensitivity. Additionally, an inhibition control assay proved to be helpful in avoiding false negative results when interpreting negative results of samples that might be of low quality or with inadequate reduction in inhibitory substances. When tested with synthetic DNA standards, the portable instrument performed at a level approaching stationary thermocyclers. In summary, the developments of suitable sample preparation methods, robust and thermal‐stable real‐time PCR assays with inhibition control, and battery‐powered portable thermocyclers with middle‐throughput offer one way forward to provide rapid, reliable molecular diagnosis under challenging field conditions.

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Section: Molecular Detection Of Asfv Dnamentioning

confidence: 99%

“…The use of a commercial battery-powered portable thermocycler (T-COR 4 TM ) for the detection of ASFV via a real-time PCR assay has previously been evaluated (174,175). In one study, reduced sensitivity of the portable thermocycler for clinical samples with very high Ct values on the gold standard qPCR was observed on a RT-PCR/PCR duplex assay for Classical and African swine fever viruses (175). In our hands, portable thermocyclers (e.g., GeneReach Pockit TM or Biomeme Franklin TM ) show comparable clinical and analytical sensitivity and specificity using ASF positive and negative experimental and field samples when compared to a laboratory thermocycler (Bio-Rad CFX 96).…”

Section: Molecular Detection Of Asfv Dnamentioning

confidence: 99%

African Swine Fever Virus: An Emerging DNA Arbovirus

et al. 2020

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African swine fever virus (ASFV) is the sole member of the family Asfarviridae, and the only known DNA arbovirus. Since its identification in Kenya in 1921, ASFV has remained endemic in Africa, maintained in a sylvatic cycle between Ornithodoros soft ticks and warthogs (Phacochoerus africanus) which do not develop clinical disease with ASFV infection. However, ASFV causes a devastating and economically significant disease of domestic (Sus scrofa domesticus) and feral (Sus scrofa ferus) swine. There is no ASFV vaccine available, and current control measures consist of strict animal quarantine and culling procedures. The virus is highly stable and easily spreads by infected swine, contaminated pork products and fomites, or via transmission by the Ornithodoros vector. Competent Ornithodoros argasid soft tick vectors are known to exist not only in Africa, but also in parts of Europe and the Americas. Once ASFV is established in the argasid soft tick vector, eradication can be difficult due to the long lifespan of Ornithodoros ticks and their proclivity to inhabit the burrows of warthogs or pens and shelters of domestic pigs. Establishment of endemic ASFV infections in wild boar populations further complicates the control of ASF. Between the late 1950s and early 1980s, ASFV emerged in Europe, Russia and South America, but was mostly eradicated by the mid-1990s. In 2007, a highly virulent genotype II ASFV strain emerged in the Caucasus region and subsequently spread into the Russian Federation and Europe, where it has continued to circulate and spread. Most recently, ASFV emerged in China and has now spread to several neighboring countries in Southeast Asia. The high morbidity and mortality associated with ASFV, the lack of an efficacious vaccine, and the complex makeup of the ASFV virion and genome as well as its lifecycle, make this pathogen a serious threat to the global swine industry and national economies. Topics covered by this review include factors important for ASFV infection, replication, maintenance, and transmission, with attention to the role of the argasid tick vector and the sylvatic transmission cycle, current and future control strategies for ASF, and knowledge gaps regarding the virus itself, its vector and host species.

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Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses

Cited by 21 publications

References 11 publications

Classical Swine Fever—An Updated Review

Classical Swine Fever—An Updated Review

Overcoming the challenges of pen‐side molecular diagnosis of African swine fever to support outbreak investigations under field conditions

African Swine Fever Virus: An Emerging DNA Arbovirus

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