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.