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

Abstract

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.

Keywords: African swine fever virus; DNA extraction; molecular detection; recombinase polymerase amplification.

Figure 1

Amplification curves of RPA (A) and real-time PCR (B). Both assays detected down to one DNA molecule per µL.

Figure 2

Limit of detection (A) and reproducibility (B) of the ASF RPA assay. Dataset of 8 RPA runs of the molecular standard 100 to 1 DNA copy/μL was used. Limit of detection is 3.5 copies/µL (A). The speed of the assay was between 5 and 7 min (B).

Figure 3

Comparison between the TT value of the RPA and Ct value of the real-time PCR. No correlation was found (R² = 0.34) as the RPA is much faster than the real-time PCR.

Figure 4

Field deployment of mobile suitcase lab in Uganda: (A) ASFV-RPA assay results of 20 samples from suspected ASF domestic pigs in Uganda. Eleven afebrile pigs tested negative, while six febrile pigs, one pig without temperature reading and two afebrile pigs tested positive. Red is animal tested positive. Black is animal tested negative. Blue thermometer indicates normal body temperature, red is pig with fever, and grey is pig with no body temperature measured. (B) Mobile suitcase lab.