Point-of-Care Testing for Sensitive Detection of the African Swine Fever Virus Genome

Abstract

African swine fever (ASF) is a contagious viral hemorrhagic disease that affects domestic pigs and wild boar. The disease is notifiable to the World Organization of Animal Health (WOAH), and causes significant deaths and economic losses. There is currently no fully licensed vaccine available. As a result, early identification of the causative agent, ASF virus (ASFV), is crucial for the implementation of control measures. PCR and real-time PCR are the WOAH-recommended standard methods for the direct detection of ASFV. However, under special field conditions or in simple or remote field laboratories, there may be no sophisticated equipment or even stable electricity available. Under these circumstances, point-of-care systems can be put in place. Along these lines, a previously published, rapid, reliable, and electricity-free extraction method (TripleE) was used to isolate viral nucleic acid from diagnostic specimens. With this tool, nucleic acid extraction from up to eight diagnostic samples can be realized in one run in less than 10 min. In addition, the possibility of completely omitting viral DNA extraction was analyzed with so-called direct real-time PCR protocols using ASFV original samples diluted to 1:40 in RNase-free water. Furthermore, three real-time PCR cyclers, developed for use under field conditions (IndiField, Liberty16 and UF-300 GenecheckerTM), were comparatively applied for the sensitive high-speed detection of ASFV genomes, with overall PCR run times between 20 and 54 min. Depending on the viral DNA extraction/releasing method used and the point-of-care cycler applied, a total time for detection of 30 to 60 min for up to eight samples was feasible. As expected, the limitations in analytical sensitivity were positively correlated to the analysis time. These limitations are acceptable for ASFV diagnostics due to the expected high ASFV genome loads in diseased animals or carcasses.

Innovative Research Offers New Hope for Managing African Swine Fever Better in Resource-Limited Smallholder Farming Settings: A Timely Update

“…However, a recent on-farm study confirmed the utility of LFD for diagnosis of ASF in clinically sick pigs [ 199 ]. Similarly, DNA extraction/releasing methods combined with different portable real-time PCR detection systems indicated that ASFV in field samples of clinically sick pigs can successfully be identified [ 207 , 208 ]. After POC testing in the field, a quick response by veterinary services will be required to ensure the community perception and acceptability of treatment or management decisions [ 194 ] for POC testing are successfully implemented.…”

Combination-Lock SlipChip Integrating Nucleic Acid Sample Preparation and Isothermal LAMP Amplification for the Detection of SARS-CoV-2

“…Nucleic acid analysis (NAA) has become an essential tool for biological research and clinical diagnostics for its high sensitivity and specificity . During the global pandemic, NAA methods, such as PCR-based methods, have become the gold standard for COVID-19 diagnosis and recovery monitoring. − Moreover, NAA has become a critical tool for the detection and investigation of numerous other infectious diseases, including tuberculosis, HIV, monkeypox, Ebola, and Zika. − In addition, NAA has emerged as a promising tool in food contamination testing and animal infectious diseases, such as Escherichia coli O157, salmonella, avian flu, and African swine fever. − …”