Clinical Validation of Two Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of African Swine Fever Virus

Xiaoxu Fan; Lin Li; Yonggang Zhao; Yutian Liu; Chunju Liu; Qinghua Wang; Yaqin Dong; Shujuan Wang; Tianying Chi; Fangfang Song; Chengyou Sun; Yingli Wang; Dengchuriya Ha; Yang Zhao; Jingyue Bao; Xiaodong Wu; Zhiliang Wang

doi:10.3389/fmicb.2020.01696

Clinical Validation of Two Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of African Swine Fever Virus

Front Microbiol. 2020 Jul 21:11:1696. doi: 10.3389/fmicb.2020.01696. eCollection 2020.

Authors

Xiaoxu Fan¹, Lin Li¹, Yonggang Zhao¹, Yutian Liu¹, Chunju Liu¹, Qinghua Wang¹, Yaqin Dong², Shujuan Wang¹, Tianying Chi¹, Fangfang Song¹, Chengyou Sun¹, Yingli Wang¹, Dengchuriya Ha³, Yang Zhao^{1

4}, Jingyue Bao¹, Xiaodong Wu¹, Zhiliang Wang¹

Affiliations

¹ National Reference Laboratory for African Swine Fever, National Surveillance and Research Center for Exotic Animal Diseases, National Surveillance and Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, Qingdao, China.
² Livestock Disease Surveillance Laboratory, China Animal Health and Epidemiology Center, Qingdao, China.
³ Vocational and Technical College, Inner Mongolia Agricultural University, Hohhot, China.
⁴ College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, China.

Abstract

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boars, and has tremendous negative socioeconomic impact on the swine industry and food security worldwide. It is characterized as a notifiable disease by World Organisation for Animal Health (OIE). No effective vaccine or treatment against ASF has so far been available. Early detection and rapid diagnosis are of potential significance to control the spread of ASF. Recombinase-based isothermal amplification assay, recombinase polymerase amplification (RPA) developed by TwistDx (Cambridge, United Kingdom) or recombinase-aided amplification (RAA) by Qitian (Wuxi, China), is becoming a molecular tool for the rapid, specific, and cost-effective identification of multiple pathogens. In this study, we aim to investigate if RPA/RAA can be a potential candidate for on-site, rapid and primary detection of ASFV. A panel of 152 clinical samples previously well-characterized by OIE-recommended qPCR was enrolled in this study, including 20 weak positive (Ct value ≥ 30) samples. This panel was consisted of different types, such as EDTA-blood, spleen, lung, lymph node, kidney, tonsil, liver, brain. We evaluated two recombinase-based isothermal amplification assays, RPA or RAA, by targeting the ASFV B646L gene (p72), and validated the clinical performance in comparison with OIE real-time PCR. Our result showed that the analytical sensitivity of RPA and RAA was as 93.4 and 53.6 copies per reaction, respectively at 95% probability in 16 min, at 39°C. They were universally specific for all 24 genotypes of ASFV and no cross reaction to other pathogens including Classical swine fever virus (CSV), Foot-and-mouth disease virus (FMDV), Pseudorabies virus, Porcine circovirus 2 (PCV2), Porcine Reproductive and respiratory syndrome virus (PPRSV). The results on detection of various kinds of clinical samples indicated an excellent diagnostic agreement between RPA, RAA and OIE real-time PCR method, with the kappa value of 0.960 and 0.973, respectively. Compared to real-time PCR, the specificity of both RPA and RAA was 100% (94.40% ∼ 100%, 95% CI), while the sensitivity was 96.59% (90.36% ∼ 99.29%, 95% CI) and 97.73% (92.03% ∼ 99.72%, 95% CI), respectively. Our data demonstrate that the developed recombinase-based amplification assay (RPA/RAA), promisingly equipped with field-deployable instruments, offers a sensitive and specific platform for the rapid and reliable detection of ASFV, especially in the resource-limited settings for the purpose of screening and surveillance of ASF.

Keywords: African swine fever virus; clinical validation; rapid isothermal amplification; recombinase aided amplification; recombinase polymerase amplification.