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Article

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

1
Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, 04103 Leipzig, Germany
2
College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University, Kampala P.O. Box 7062, Uganda
3
National Agricultural Research Organisation, Entebbe P.O. Box 295, Uganda
4
Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, 17493 Greifswald, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Manuel Borca
Viruses 2021, 13(9), 1731; https://doi.org/10.3390/v13091731
Received: 29 July 2021 / Revised: 23 August 2021 / Accepted: 26 August 2021 / Published: 31 August 2021
(This article belongs to the Special Issue African Swine Fever Virus 2021)
African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures. View Full-Text
Keywords: African swine fever virus; recombinase polymerase amplification; DNA extraction; molecular detection African swine fever virus; recombinase polymerase amplification; DNA extraction; molecular detection
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MDPI and ACS Style

Ceruti, A.; Kobialka, R.M.; Ssekitoleko, J.; Okuni, J.B.; Blome, S.; Abd El Wahed, A.; Truyen, U. Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay. Viruses 2021, 13, 1731. https://doi.org/10.3390/v13091731

AMA Style

Ceruti A, Kobialka RM, Ssekitoleko J, Okuni JB, Blome S, Abd El Wahed A, Truyen U. Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay. Viruses. 2021; 13(9):1731. https://doi.org/10.3390/v13091731

Chicago/Turabian Style

Ceruti, Arianna, Rea Maja Kobialka, Judah Ssekitoleko, Julius Boniface Okuni, Sandra Blome, Ahmed Abd El Wahed, and Uwe Truyen. 2021. "Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay" Viruses 13, no. 9: 1731. https://doi.org/10.3390/v13091731

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