目录
摘要Ⅱ
关键词Ⅱ
AbstractⅡ
引言
引言1
1材料与方法3
1.1病毒株和病毒3
1.2标准质粒DNA制备 3
1.3引物设计 3
1.4 DNA、RNA提取和cDNA合成3
1.5PCR试纸条的制备4
1.6 优化新型PCR检测5
1.7 PCRLFS检测程序5
1.8 敏感性评估5
1.9 特异性评估5
1.110模拟样品的检测和统计分析5
2结果与分析5
2.1 验证标准质粒中的目标片段5
2.2 PCR程序的优化6
2.3 PCRLFS检测的敏感性7
2.4 PCRLFS检测的特异性8
2.5模拟样品检测和统计分析9
3讨论 10
致谢11
参考文献12
非洲猪瘟病毒PCR试纸条快速检测方法的构建
摘 要
非洲猪瘟（ASF）是一种由非洲猪瘟病毒（ASFV）在家猪和野猪中引起的高度传染性疾病，在中国造成巨大的经济损失。由于其极强的感染力，迫切需要建立一种方便，低成本的诊断方法，以现场快速检测病毒，及时实施控制措施。本研究针对主要结构蛋白p72基因的聚合酶链反应（PCR）与侧向流动条（LFS）相结合，用于ASFV的快速检测。 PCRLFS方法检测非洲猪瘟病毒与其他猪病毒无交叉反应。该检测方法最低能检测15个拷贝 的ASFV质粒模板。在300份加标准质粒样品的临床诊断中，通过PCRLFS检测，OIE验证的实时荧光定量PCR检测和市售试剂盒诊断ASFV的阳性率分别为36.33％（109/300），26.67％（80 / 300）和37.33％（112/300）。 PCRLFS检测和OIE验证的实时荧光定量PCR检测的符合率为89.67％（269/300），kappa值为0.763（p ＜0.001），PCRLFS和商业试剂盒的符合率为97.67％（ 293/300），kappa值为0.950（p ＜0.001）。从样品处理到结果报告的整个过程可以在1 *51今日免费论文网|www.51jrft.com +Q: #351916072# 
小时内完成。结果表明，已建立的PCRLFS检测方法适用于ASFV的现场检测。
RAPID ONSITE DETECTION OF AFRICAN SWINE FEVER VIRUS USING POLYMERASE CHAIN REACTION WITH A LATERAL FLOW STRIP
ABSTRACT
African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV) in domestic pigs and wild boars, which results in huge economic losses in China. Because of its extremely infectious capacity, there is an urgent need to establish a convenient and lowcost diagnostic method for rapid and onsite detection of the virus to timely implement the control measures. In this study, a polymerase chain reaction (PCR) in combination with lateral flow strip (LFS), targeting the major structural protein p72 gene, was established for rapid detection of ASFV. The PCRLFS assay displayed no crossreactivity to other swine viruses. The sensitivity of this assay was 15 copies/reaction of ASFV plasmid template. In clinical diagnoses of 300 plasmidspiked samples, the positive rate of the diagnosis of ASFV by PCRLFS assay, OIEvalidated realtime PCR assay, and commercial kit reached 36.33% (109/300), 26.67% (80/300) and 37.33% (112/300) respectively. The coincidence rate of PCRLFS assay and OIEvalidated realtime PCR assay was 89.67% (269/300), with a kappa value of 0.763 (p＜0.001) and that of PCRLFS and commercial kit was 97.67% (293/300), with a kappa value of 0.950 (p＜0.001). The entire procedure, from specimen processing to result reporting, can be completed within 1 hr. Our results demonstrated that the established PCRLFS assay is suitable for onsite detection of ASFV.
KEY WORDS：African swine fever virus；PCR, Lateral flow strip；Simulative samples；Onsite detection

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