Introduction
African swine fever (ASF) is an extremely contagious disease of wild
boar and domestic pigs with an almost 100% mortality rate, causing
significant economic trauma to the pig industry in affected countries
(Dixon et al., 2020). The clinical symptoms of ASF infection can be
manifested from subclinical infection to sudden death with few other
signs. This fact, together with the great similarities of clinical
presentations and lesions between ASF and other hemorrhagic pig diseases
(Dixon et al., 2020; Schulz et al., 2017), make differential laboratory
diagnosis compulsory. Since there is no vaccine commercially available
as of 2020 (Revilla et al., 2018; Teklue et al., 2020), control and
eradication strategies are timely and comprehensive culling of infected
pigs, relying on accurate and rapid laboratory diagnostic screening of
ASFV-positive or suspected cases before the large-scale outbreak (Arias
et al., 2018).
Currently, molecular methods are recommended for the diagnosis and
containment of ASFV (Fernandez-Pinero et al., 2013; OIE, 2018; Wang et
al., 2020; Ye et al., 2019). Conventional PCR methods which require
post-amplification manipulation (Aguero et al., 2003; OIE, 2018) are
being broadly replaced by the real-time PCR system. Significantly, the
World
Organisation for Animal Health (OIE) -recommended real-time qPCR method
amplifies a viral DNA fragment of 250 bp long (King et al., 2003; OIE,
2018), which is considered now as non-optimal size for a real-time PCR
system (Fernandez-Pinero et al., 2013). Although various real-time PCR
methods give good sensitivity and specificity rates, the robustness of
the methods are decreased when weak ASFV-positive samples are analyzed
(Gallardo et al., 2019).
One of the bottlenecks for PCR-based assays to detect DNA targets from
clinical samples is that the presence of inhibitors suppresses the
activity of DNA polymerase, necessitating a DNA purification prior to
amplification. Various methods for DNA extraction have been developed
(Thatcher, 2015). However, these methods are generally labor-intensive
and time-consuming. Furthermore, the multiple sample processing steps
involved in these methods increase the risk of cross-contamination and
human error, making them sub-optimal for high-throughput application and
widespread deployment (Fernandez-Pinero et al., 2013; King et al., 2003;
Wang et al., 2020). The “direct PCR” was then developed to make the
analysis method less sensitive to interference by using mutants ofTaq DNA polymerase that are more resistant to inhibitors from
complex sample backgrounds so that the preparation procedure could be
bypassed (Kermekchiev et al., 2009; Leelawong et al., 2019; Li et al.,
2019; Zhang et al., 2010), but sometimes at the expense of removing the
exonuclease activity required for cleavage of hydrolysis probes and
increasing
cost (Leelawong et al., 2019). PCR can also be optimized through the use
of various PCR enhancer cocktail (Li et al., 2019; Zhang et al., 2010)
and specific PCR buffers (Bu et al., 2008; Liu et al., 2018) to reduce
the inhibitory effect of blood components. However, the application of
these methods in practice is challenging as the fluorescent signal is
quenched by compounds such as heme and hemoglobin (Kang et al., 2014),
so that only a very small volume of sample can be assayed directly, and
the sensitivity is often compromised compared with using equivalent
amount of purified DNA. Automated extraction instrument provides another
potential solution (Flannery et al., 2020), but significantly increasing
the assay cost.
Herein, we describe the development of a specific high-sensitivity
multiple-probe-assisted DNA capture and amplification technology
(MADCAT) for direct detection of African swine fever virus DNA molecule
in blood or tissue homogenates without DNA extraction to effectively
overcome the challenges mentioned above.