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.