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Specific High-sensitivity Multiple-probe-assisted DNA Capture and Amplification Technology for Direct Detection of African Swine Fever Virus without DNA Extraction
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  • Huicong Wang,
  • Hongru Pian,
  • Lihua Fan,
  • Jian Li,
  • Jifei Yang,
  • Zhi Zheng
Huicong Wang
Peking Union Medical College School of Basic Medicine
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Hongru Pian
Peking Union Medical College School of Basic Medicine
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Lihua Fan
Peking Union Medical College School of Basic Medicine
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Jian Li
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences
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Jifei Yang
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences
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Zhi Zheng
Peking Union Medical College School of Basic Medicine
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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.