Here we develop MADCAT for high-throughput, large-scale ASFV screening application, which is based upon a series of capture probes hybridizing alternate regions on each strand of the target DNA (Figure 1A). Initially we used our previously established method for capturing single-stranded RNA to capture the denatured DNA (Xu & Zheng, 2016). However, this single-strand capture method used in DNA capture was shown to have a low capture efficiency (Figure 1B), probably due to the competitive interference of the complementary strand of the target DNA. In this study, two other kinds of capture probes were used, each targeting both DNA strands with a multiple-probe-per-strand design (Figure 1A, B). By simultaneously capturing both strands they achieved near 100% capture efficiency (Figure 2B). It is possible the steric hindrance after probe binding makes it difficult for the two stands to reanneal. Compared with another capture-based biosensor method, which exploited a triplex formation with ssDNA/LNA chimeric probes (Biagetti et al., 2018), the MADCAT approach does not require modified nucleotide for the probes and will not be affected by potential blood impurities, which may produce non-specific responses and non-interpretable results in biosensor method. In addition, the simultaneous action of multiple capture probes ensures that all ASFV genotypes can be captured, tolerating genetic drift or point mutations.
Notably, unlike any current molecular technology, which extracts all the DNA into a solution followed by selective amplification, our method specifically captures the ASFV target on the solid support, and can completely separate the target from irrelevant sequences, impurities and inhibitors through a simple washing step, leaving the target DNA as the only amplifiable template in the subsequent PCR reaction. This can not only reduce non-specific signal interference, but also increase the efficiency of amplification, and can even test pooled samples in large-scale screening without losing sensitivity. When spiked plasmid DNA in porcine blood was tested, the Ct value was almost identical to that of DNA without blood, and the same LOD was observed (Figure 2B), demonstrating a much better ability against blood interference than most “direct PCR” methods. By circumventing the extraction processes, DNA loss and laboratory personnel error may be minimized, and overall sample processing time and cost can be reduced. Due to reduced steps, this method not only saves labor, but also increases the assay reproducibility with a coefficient of Ct value variation (C.V) < 3% (Table 1). Despite the additional wash step, our method can handle high-concentration samples without any cross-contamination (Figure 3), probably because the target DNAs are bound at the bottom of the well not in the solution during the handling. Considering that only 12 μL of sample input is required, the MADCAT method greatly reduces the need for sample volume and eases sampling pressure, whereas a DNA extraction workflow often consumes 200 μL of precious samples (Aguero et al., 2003; Gallardo et al., 2015; King et al., 2003; OIE, 2018). Although less sample volume is required, this method amplifies more sampled DNA than extraction-based method, which can only amplify a portion of the extracted DNA in the sampled volume.
Since the routine virus testing of infected pigs has become a key element of control strategy for ASF (Dixon et al., 2020), the high-throughput MADCAT has a potential to meet the needs of active surveillance system with its ELISA-like workflow on 96-well plate format. Throughout the procedure only one plate was used, and 96 samples can be easily assayed with minimal user input and a sample-to-result time within 100 min (Figure 1 C). Coupled with an ultra-high sensitivity of 0.5 DNA copies/μL or 6 DNA copies/reaction, MADCAT offers the potential of large-scale epidemiological screening, with sample pooling strategy, for early ASFV infections or even asymptomatic infections.
In conclusion, we have developed a specific high-sensitivity, easy to use, inexpensive, and accurate molecular assay suitable for high-throughput ASFV screening. This new DNA detection platform holds great application potential in detecting other types of nucleic acid targets, such as other infectious pathogens and disease genetic markers.