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

Arianna Ceruti,Julius Boniface Okuni,Ahmed Abd El Wahed,Judah Ssekitoleko,Rea Maja Kobialka,Sandra Blome,Uwe Truyen

doi:10.3390/v13091731

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.

Highlights

African swine fever causes a highly lethal, contagious disease in pigs, threatening the global swine industry and national economies
52 whole blood samples from experimentally infected domestic pigs were used in the study
The B604L gene was used as the target for the developed recombinase polymerase amplification (RPA) assay

Summary

Introduction

African swine fever causes a highly lethal, contagious disease in pigs, threatening the global swine industry and national economies. The virus is circulating in a sylvatic cycle among African wild suids (mainly Warthogs, Phacochoerus africanus) and Ornithodoros soft ticks in sub-Saharan Africa. This cycle is not accompanied by overt disease [10]. The most common form is the acute infection that induces high fever, lethargy, respiratory and digestive dysfunctions (often with hemorrhagic tendency), abortion, and sudden deaths. Since it shows great similarities with other infectious diseases regarding clinical and pathological pictures (e.g., classical swine fever (CSF)) [19], differential laboratory diagnosis is essential. Since neither effective treatment nor vaccination are available, the most essential control measures are identification of infected animals in wild or domestic pigs and immediate culling and movement restriction

Objectives

Methods

Results