非洲猪瘟实时荧光RPA诊断方法建立及应用

[目的]非洲猪瘟(African swine fever,ASF)自2018年首次暴发于我国沈阳后,迅速扩散至全国,对我国养猪业造成了沉重打击.研究针对其病原非洲猪瘟病毒(African swine fever virus,ASFV)建立一种快速核酸检测技术,为及时发现、准确处理ASF疫情提供一种快速诊断方法.[方法]...

Development and Application of a Real-Time Fluorescent RPA Diagnostic Assay for African Swine Fever

【Objective】 After the first outbreak of African Swine Fever (ASF) in Shenyang, China in 2018, it has rapidly spread to the whole country, severely hitting the pig industry. This study aimed to establish an optimized nucleic acid testing technique for African Swine Fever Virus (ASFV), so as to provide a fast and accurate method for early diagnosis and accurate treatment of ASF outbreaks. 【Method】 Appropriate primers and probes were designed and screened for the conserved gene B646L (p72) of ASFV, and a real-time fluorescent RPA assay based on recombinase polymerase amplification (RPA) was established. The reaction system, reaction conditions and sample treatment steps were optimized. Specificity and sensitivity of the optimized detection method were evaluated by using quality controls. In addition, 1 009 clinical samples were tested by the optimized real-time RPA, after which the results were further confirmed by the real time PCR recommended by OIE and through virus isolation. 【Result】 A pair of primers-probe combinations was successfully screened, and a real-time fluorescence RPA for detection of ASFV p72 gene was developed. The total volume of optimized reaction system was 25 μL. The reaction conditions were set as 39℃ 10 s, 39℃ 20 s, 40 cycles on the fluorescence quantitative PCR instrument, and the whole amplification reaction needs about 20 min. The analysis method at room temperature could replace the traditional nucleic acid extraction method, thus the whole process of sample treatment, nucleic acid amplification and result reading could be completed in 30 min. Specific evaluation showed that the real-time RPA was negative for porcine parvovirus (PPV), pseudorabies virus (PRV), circovirus type1/2 (PCV1/2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV); the sensitivity evaluation showed that the assay could detect type I/II/IX ASFV samples, and could detect 10 copies/μL of ASFV positive simulated blood samples and 1﹕10^3.0 dilution of positive clinical samples, which was as sensitive as the OIE-recommended qPCR method. Seventeen out of 1 009 clinical samples were tested positive using the real-time RPA, with the same results as by qPCR, 17 positive cultures were obtained from virus isolation. 【Conclusion】 A real-time RPA diagnostic method for ASF was developed, which was proved to be simple, less time consuming with high sensitivity and specificity, providing a new, simple, specific and rapid diagnostic method for ASF.