非洲猪瘟间接ELISA诊断试剂盒的研究

用带有编码非洲猪瘟病毒衣壳蛋白Ｐ７２ 基因的重组杆状病毒（Ｂａｃｐ７２）作载体，在ｓｆ９细胞中表达并得到重组Ｐ７２蛋白，ＳＤＳ－ＰＡＧＥ可得到分子量在 ７２ｋＤａ左右的电泳带。用标准阳性非洲猪瘟血清对 Ｐ７２蛋白进行 ＥＬＩＳＡ检测，证明该蛋白具有生物学活性。用Ｐ７２作为间接法的包被抗原，对ＥＬＩＳＡ反应条件进行了优化。确定最佳包被液为ＰＢＳ（ｐＨ７．２）、最佳封闭液为１％ＰＣＴ、最佳血清稀释液为４％ＰＥＧ６０００／ＰＢＳ、最佳冲洗液为０．５Ｍ ＮａＣｌ／０．５％Ｔｗｅｅｎ－２０／ＰＢＳ（ｐＨ７．２）。本实验反应体系采用５０μｌ的微量法，可节约试剂及抗原。反应在２小时内即可完成，达到了快速诊断的目的。包被了抗原并用封闭液封闭后的酶标板密封后保存于－２０℃的冰箱中，至少可以保存５个月。阻断试验和交叉试验表明ＥＬＩＳＡ法有良好的特异性。间接ＥＬＩＳＡ比Ｄｏｔ－ＥＬＩＳＡ法具有更高的灵敏性。血清学调查没有得到阳性结果，与我国实际情况相符。用Ｂａｃｐ７２表达的非洲猪瘟病毒Ｐ７２蛋白抗原作为间接ＥＬＩＳＡ的检测抗原来检测非洲猪瘟血清具有快速、简单、无感染的特点。本实验为非洲猪瘟ＥＬＩＳＡ检测试剂盒的最终组装提供了实验依据。  

非洲猪瘟病毒环介导等温扩增快速检测方法的建立

本试验利用环介导等温扩增技术（LAMP）建立了一种快速高效检测非洲猪瘟病毒的方法。整个反应在水浴锅中恒温进行，不需要其他昂贵仪器，30 min即可得到阳性结果，灵敏度是OIE标准PCR方法的100倍，并且与其他常见猪DNA病毒无交叉反应。该方法非常适合在野外现场或缺乏试验条件的边远地区检测非洲猪瘟病毒。  

用PCR技术快速检测非洲猪瘟病毒

选择编码非洲猪瘟病毒结构蛋白ＶＰ７３的部分基因片段为模板，用ＰＣＲ技术检测非洲猪瘟病毒ＤＮＡ，电泳分离可见１条长约１ｋｂ的ＤＮＡ扩增带。本方法具有安全、快速、敏感等特点  

一种用于非洲猪瘟病毒检测的PCR方法

基于非洲猪瘟病毒(ASFV)VP72基因设计引物,建立一种检测非洲猪瘟病毒的PCR方法。应用本研究所建立的方法与OIE参考的方法进行比较,并对参考实验室提供的非洲猪瘟病毒17个分离株的基因组以及本实验室收集的野外样品进行检测。结果显示:本研究设计的引物具有良好的特异性,与猪的其他病原没有交叉反应;其敏感性与OIE参考的方法相当;所建立的PCR方法能够成功扩增非洲猪瘟病毒17个分离株的基因组,野外样品检测均为阴性。根据上述研究结果,本研究所建立的方法具有很好的应用性,能够用于非洲猪瘟疫病的诊断以及防控。  

环介导恒温扩增技术快速检测非洲猪瘟病毒

以人工合成的非洲猪瘟病毒（ASFV）P72全长基因为模板，利用在线软件Primer Explorer4设计环介导恒温扩增（LAMP）的内、外引物，通过对反应体系和反应条件进行优化，建立了ASFV的LAMP检测方法，同时进行了敏感性和特异性试验。结果表明，所建立的LAMP检测方法最低检测限可达10拷贝质粒DNA，且具有良好的特异性，LAMP产物的酶切鉴定也验证了反应的特异性。对凝胶电泳后紫外观察、肉眼观察颜色变化、生成沉淀观察以及实时荧光PCR扩增曲线Ct值判定等不同判定方法进行比较表明，以上结果判定方法各有其优缺点，其中荧光PCR的敏感性最高，染色法相对较方便直观，电泳法敏感性稍低。  

非洲猪瘟的研究进展

非洲猪瘟是猪的一种急性、高度接触传染性疾病。其感染率高, 多种传播途径,死亡率可达100％,现还无有效的疫苗及治疗措施。非洲猪瘟在临床上与猪瘟相似，虽这2种疾病的病原体完全不同，但在诊断时极易误诊，为了进一步了解该病,作者综述了该病的病原、流行病学、临床症状、鉴别诊断及我国现状，从而为诊断该病提供了依据。  

Temporal and spatial patterns of African swine fever in Sardinia

Temporal patterns and spatial distribution of African swine fever (ASF) were studied through the analysis of routinely collected data in the ASF-endemic area of the Province of Nuoro, Sardinia. During 1993–1996, ASF outbreaks were reported from 45 out of the 82 municipalities of the study area. Overall farm-level incidence rate (IR) was 1.3 outbreaks per 100 farms-year. ASF peaked in 1995 (IR=1.8) and declined in 1996 (IR=0.82). Significant (P<0.05) spring peaks of ASF outbreaks and affected municipalities were detected using statistical methods for circular distributions. Spatial clustering of ASF-affected municipalities, as evaluated by join-count statistics, was significant in 1993 (Z_jc=−3.0, P<0.01) and 1994 (Z_jc=−3.2, P<0.01) but not in 1995 (Z_jc=−0.6, P=0.55) and 1996 (Z_jc=−1.2, P=0.23). Extensive pig farming and ASF were spatially co-distributed (κ=0.51, 95% CI=0.33–0.70).  

The lesional changes and pathogenesis in the kidney in african swine fever

African swine fever is a viral haemorrhagic disease of pigs which has been used as a model for the study of viral haemorrhagic diseases in man. The acute course of the disease is characterized by acute proliferative glomerulonephritis, with viral replication in mesangial cells and occasional focal necrosis of the renal tubular system; hyperplasia of the collecting ducts is associated with evident virus replication. Haemorrhages have been attributed to endothelial dysfunction, aggravated by virus replication in endothelial cells in the final stages of the disease. The renal interstitium displays intense oedema and an infiltrate largely composed of macrophages. Virus replication has also been observed in fibroblasts and in the smooth0muscle cells of arterioles and venules.In subacute-chronic forms of the disease, various types of glomerulonephritis are observed, ranging from mesangial proliferative glomerulonephritis to focal and segmental hyalinosis associated with immune-mediated phenomena. No striking changes are reported in the renal tubular system. Interstitial haemorrhages are associated with diapedesis due to immunologically mediated events. The interstitium has also been found to contain a lymphohistiocytic infiltrate with abundant plasma cells. No evidence has been reported of viral replication in any cell population.Abbreviations ASF African swine fever - ASFV African swine fever virus - C complement component - DNA deoxyribonucleic acid - HAD₅₀ 50% haemoadsorbing doses - Ig immunoglobulin - MPS mononuclear phagocytic system - PAS periodic acid-Schiff reagent