Overcoming the challenges of pen‐side molecular diagnosis of African swine fever to support outbreak investigations under field conditions
- 16 December 2018
- journal article
- research article
- Published by Hindawi Limited in Transboundary and Emerging Diseases
- Vol. 66 (2), 908-914
- https://doi.org/10.1111/tbed.13103
Abstract
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, dry‐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 a dry‐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 of inhibitory substances. When tested with synthetic DNA standards, the portable instrument performed at a level approaching the stationary thermocycler. In summary, the developments of suitable sample preparation, 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.This publication has 19 references indexed in Scilit:
- Molecular Characterization of African Swine Fever Virus, China, 2018Emerging Infectious Diseases, 2018
- Identification of Wild Boar–Habitat Epidemiologic Cycle in African Swine Fever EpizooticEmerging Infectious Diseases, 2018
- Quantitative assessment of social and economic impact of African swine fever outbreaks in northern UgandaPreventive Veterinary Medicine, 2017
- Reemergence of African Swine Fever in Zimbabwe, 2015Emerging Infectious Diseases, 2017
- Identification of a New Genotype of African Swine Fever Virus in Domestic Pigs from EthiopiaTransboundary and Emerging Diseases, 2016
- African swine fever in wild boar in Europe: a notable challengeVeterinary Record, 2015
- Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the fieldJournal of Virological Methods, 2014
- Molecular Diagnosis of African Swine Fever by a New Real-Time PCR Using Universal Probe LibraryTransboundary and Emerging Diseases, 2012
- Detection of African swine fever virus by loop-mediated isothermal amplificationJournal of Virological Methods, 2010
- Genetic characterisation of African swine fever viruses from outbreaks in southern Africa (1973–1999)Veterinary Microbiology, 2007