美洲型猪繁殖与呼吸综合征病毒实时荧光重组酶聚合酶扩增技术(RPA)检测方法的建立及其应用

Development and Application of A Real-time RPA Method for Detection of American Genotype of Porcine Reproductive and Respiratory Syndrome Virus

美洲型猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)在我国出现了多种基因亚型毒株混合流行和重组的复杂变化,其引起的猪繁殖与呼吸综合征(PorcineReproductiveand Respiratory Syndrome,PRRS)已成为危害程度仅次于非洲猪瘟的重要疫病。为建立一种快速而敏感性高的美洲型PRRSV检测方法,本研究分析了国内美洲型全基因组序列,利用重组酶聚合酶扩增技术(Recombinase Polymerase Amplification,RPA),设计合成特异性引物和探针,优化反应体系和条件,建立美洲型PRRSV实时荧光RPA检测方法,通过特异性、敏感性、重复性试验及田间样品的比对检测,评价该方法的检测能力,并测定和分析了田间阳性样品的ORF5基因序列。结果显示,该方法在42℃、20 min内即可完成检测,最低检测美洲型PRRSV载量为50拷贝/μL,且与欧洲型PRRSV、口蹄疫病毒、猪瘟病毒、塞内卡病毒、非洲猪瘟病毒、猪伪狂犬病毒、猪圆环病毒、猪流行性腹泻病毒无交叉反应,特异性好;检测50份田间样品该方法的阳性检出率为34%,与实时荧光RT-PCR检测结果的符合率为100%;ORF5序列分析显示检出阳性样品中NADC30-Like毒株占比为88%。结果表明,本研究建立的美洲型PRRSV实时荧光RPA检测方法特异性强、敏感性高,满足PRRSV的快速诊断和流行病学调查。

American genotypes of the porcine reproductive and respiratory syndrome virus(PRRSV) have emerged in China as a complex variation of a mixed epidemic and recombination of multiple genetic subtypes of the virus. Porcine reproductive and respiratory syndrome(PRRS) has become an important disease which is second to African swine fever in terms of hazard level. We wished to establish a rapid and sensitive method for PRRSV detection. The whole genome sequence of American-genotype strains in China in recent years were analyzed. Specific primers were designed and synthesized, and a real-time recombinase polymerase amplification(RPA) test method established by optimizing the reaction system and conditions. Tests for specificity, sensitivity, and repeatability were carried out to compare the detection ability of our method with those of realtime reverse transcription-polymerase chain reaction(RT-PCR). The open reading frame(ORF)5 gene sequences of field-positive samples were determined and analyzed. Our method could be completed in 20 min at 42°C. The minimum viral load for detecting strains of North American genotypes was 50 copies/μL. There was no cross-reactivity with the foot-and-mouth disease virus, classical swine fever virus, Senecavirus A, African swine fever virus, porcine pseudorabies virus, porcine circovirus, or porcine epidemic diarrhea virus. The percent positive detection of our method for 50 field samples was 34%, and compliance with the results of realtime RT-PCR was 100%. The percentage of NADC30-like strains in positive samples according to analyses of the ORF5 sequence was 88%. Our results indicated that the real-time RPA assay for American genotypes of the PRRSV we established had strong specificity and high sensitivity. Our method could be used for the rapid diagnosis and epidemiological investigation of the PRRSV.