Development of a novel lateral flow assay for detection of African swine fever in blood

Abstract

BackgroundAfrican swine fever (ASF) is a viral infectious disease of domestic and wild suids of all breeds and ages, causing a wide range of hemorrhagic syndromes and frequently characterized by high mortality. The disease is endemic in Sub-Saharan Africa and Sardinia. Since 2007, it has also been present in different countries of Eastern Europe, where control measures have not been effective so far. The continued spread poses a serious threat to the swine industry worldwide. In the absence of vaccine, early detection of infected animals is of paramount importance for control of the outbreak, to prevent the transmission of the virus to healthy animals and subsequent spreading of the disease. Current laboratory diagnosis is mainly based on virological methods (antigen and genome detection) and serodiagnosis.ResultsIn the present work, a Lateral Flow Assay (LFA) for antigen detection has been developed and evaluated. The test is based on the use of a MAb against VP72 protein of ASFV, the major viral capsid protein and highly immunogenic. First experiments using VP72 viral and recombinant protein or inactivated culture virus showed promising results with a sensitivity similar to that of a commercially available Antigen-ELISA. Moreover, these strips were tested with blood from experimentally infected pigs and field animals and the results compared with those of PCR and Antigen-ELISA. For the experimentally infected samples, there was an excellent correlation between the LFA and the ELISA, while the PCR always showed to be more sensitive (38 % positive samples by PCR versus 27 % by LFA). The LFA was demonstrated to be positive for animals with circulating virus levels exceeding 104 HAU. With the field samples, once again, the PCR detected more positives than either the Antigen-ELISA or LFA, although here the number of positive samples scored by the LFA exceeded the values obtained with the Antigen-ELISA, showing 60 % positivity vs 48 % for the ELISA. For the two groups of sera, the specificity was close to 100 % indicating that hardly any false positive samples were found.ConclusionsThe newly developed LFA allows rapid and reliable detection of ASFV, at field and laboratory level, providing a new useful tool for control programs and in situations where laboratory support and skilled personnel are limited.

Paper‐based point‐of‐care testing for cost‐effective diagnosis of acute flavivirus infections

“…Fiber glass paper is located upstream from the conjugate pad (made of cellulose paper) and the test strip (nitrocellulose membrane). 20 Conversely, in the present study, the analytical device that has been developed consists of a single piece of fiber glass paper. The proof of concept, using this microfluidic device, has been performed using the NS1 proteins of dengue and Zika as target antigens to be detected from plasma and whole blood samples, in simplex and multiplex formats.…”

“…A piece of fiber glass paper is used to separate red blood cells from plasma. Fiber glass paper is located upstream from the conjugate pad (made of cellulose paper) and the test strip (nitrocellulose membrane) . Conversely, in the present study, the analytical device that has been developed consists of a single piece of fiber glass paper.…”

Gaps in African swine fever: Analysis and priorities

“…New technologies including lateral flow devices (pen‐side tests) and portable PCR machines that allow rapid diagnosis have been recently developed (Sastre, Gallardo, et al., ; Sastre, Pérez, et al., ). A deeper validation under field conditions should be encouraged.…”

“…and portable PCR machines that allow rapid diagnosis have been recently developed (Sastre, Gallardo, et al, 2016;Sastre, P erez, et al, 2016). 2015).…”

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

“…Both quantitative PCR (qPCR) and conventional PCR are fast and sensitive methods for detecting ASFV 9,[11][12][13] . However, PCR methods require skilled operation and thermocyclers, making them less suitable for the field applications 14 . Isothermal amplification allows for easier operation and rapid amplification of DNA at a constant temperature independent of lab instruments 8,[15][16][17][18] .…”