【摘要】 为建立欧亚类禽H1亚型猪流感病毒（EA H1N1 SIV）的快速检测方法，本研究根据EA H1N1 SIV HA基因保守区序列，采用Primer5.0软件设计特异性引物和探针，采用矩阵法优化各反应条件后建立了检测EA H1N1 SIV的荧光定量RT-PCR(RT-qPCR)方法。采用本研究建立的Taq Man RT-qPCR方法分别检测EA H1N1 SIV、甲型H1亚型SIV、经典型H1亚型SIV、H3亚型SIV、猪瘟病毒、高致病性猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒、猪轮状病毒，评估该方法的特异性；以1×10~2拷贝/μL～1×10~8拷贝/μL的质粒标准品p MD18-T-HA作为模板，采用本研究建立RT-qPCR扩增，评估该方法的敏感性；分别以1×10~3拷贝/μL～1×10~5拷贝/μL的质粒标准品作为模板，利用该RT-qPCR方法在同一时间和不同时间检测，进行重复性试验，评估该方法的重复性。结果显示，该方法仅能检测到EA H1N1SIV，其他猪常见病毒均为阴性；对重组质粒标准品的检测限为1×10~2拷贝/μL；批内和批间重复性试验的变异系数CV值均小于5%。采用该RT-qPCR方法及常规病毒分离鉴定方法同时检测100份临床阴性猪鼻拭子样品和130份病毒分离鉴定为EA H1N1 SIV阳性的猪鼻拭子临床样品。结果显示该RT-qPCR方法对130份猪鼻拭子阳性样品均检测为阳性，对100份临床样品均检测为阴性，与病毒分离鉴定方法的符合率达100%，上述结果表明，本研究建立的Taq Man RTqPCR方法特异性强、敏感性高、重复性与稳定性较好，为EA H1N1 SIV临床样品的检测及流行病学调查提供快速便捷的技术支持。更多还原

【Abstract】 To establish a rapid detection method for Eurasian avian-like(EA) H1 swine influenza virus(EA H1N1 SIV),specific primers and a probe were designed targeting the conserved region of the EA H1N1 SIV HA gene using Jalview software.After the optimization of various reaction conditions, a Taq Man fluorescent quantification reverse transcription PCR(RTqPCR) assay for detecting the EA H1N1 subtype SIV was developed. To evaluate the specificity of the assay, the established RTqPCR method was used to detect EA H1N1 SIV, influenza A H1 subtype SIV, classical H1 subtype SIV, H3 subtype SIV, classical swine fever virus(CSFV), highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV), porcine transmissible gastroenteritis virus(TGEV), and porcine rotavirus(PoRV). The sensitivity of the assay was assessed using recombinant plasmid standards(pMD18-T-HA) at concentrations ranging from 1 × 10~2 copies/??L to 1 × 10~8 copies/??L as templates. To evaluate reproducibility, intra-reproducibility assay and inter-reproducibility assay were performed using plasmid standards at concentrations ranging from 1×10~3 copies/??L to 1×10~5 copies/??L. The results demonstrated that the assay was highly specific, exclusively detecting EA H1N1 SIV while yielding negative results for other common swine viruses. The limit of detection(LOD) for the recombinant plasmid standard was 1×10~2 copies/μL. The coefficients of variation(CVs) for both intra-repetitive assay and inter-repetitive assay were below 5%. Furthermore, the established RT-qPCR assay and conventional virus isolation methods were simultaneously applied to test 100 clinical negative porcine nasal swab samples and 130 porcine nasal swab samples previously confirmed positive via chicken embryo isolation. The RT-qPCR assay successfully identified all 130 positive nasal swab samples and yielded negative results for the 100 uninfected clinical samples, demonstrating a 100% concordance rate with the traditional virus isolation assay. In conclusion, the Taq Man RT-qPCR assay developed in this study exhibits strong specificity, high sensitivity, and excellent reproducibility and stability. It provides a rapid and convenient technical foundation for the clinical detection and epidemiological surveillance of EA H1N1 SIV.更多还原