Specific High-sensitivity Multiple-probe-assisted DNA Capture and
Amplification Technology for Direct Detection of African Swine Fever
Virus without DNA Extraction
Abstract
African Swine Fever (ASF) is one of the most devastating infectious
diseases affecting domestic pigs and wild boar. The grave socio-economic
impact of African Swine Fever infection at a global level makes
large-scale rapid and robust diagnosis a critical step towards effective
control. However, the nucleic acid purification required in most
molecular detection methods is time- and labor-intensive, prone to
nucleic acid loss or contamination, and impractical for massive active
screening or for use in resource-limited areas. Here we describe
multiple-probe-assisted DNA capture and amplification technology
(MADCAT) - a novel sensitive, simple, and reliable method for detecting
ASFV directly from whole blood or other complex matrices. Through the
unique DNA capture method which specifically capture only the target DNA
onto the well for subsequent amplification, MADCAT abandons the
complicated extraction protocol and achieves ultrafast and
high-throughput detection. The sample-to-result time for 96 samples is
about 100 min, as compared with the 3 - 4 h time of the standard real
time qPCR method. The limit of detection (LOD) is 0.5 copies/μL and is
10 times more sensitive than an OIE-recommended qPCR assay when testing
serially diluted whole blood samples. The assay is 100% specific
against other common swine pathogens. In clinical diagnosis of 48 field
samples, all 22 positive samples were correctly identified with lower Ct
values than OIE-recommended qPCR, confirming its high diagnostic
sensitivity (100%). Owing to its high-throughput, specific
high-sensitivity, and cost-efficient features, MADCAT shows great
potential for future use in clinical ASFV active screening.