摘要：本研究根据从NCBI数据库中下载到的84条DHAV-1的VP1基因核酸序列的比对结果，设计出了一对可特异性扩增140bp片段的上、下游引物及相应的TaqMan探针，通过对引物浓度、探针浓度及退火温度的优化，建立了Ⅰ （甲）型鸭肝炎病毒1系（DHAV-1）的实时荧光定量RT-PCR检测方法，并对该方法的特异性、敏感性和重复性进行了分析。结果表明，该检测方法呈现出典型的S型扩增曲线，能扩增出目的条带，其敏感性能达到1×102拷贝，批内重复及批间重复的变异系数均小于5%。同时特异性分析表明，本方法对其他七种常感染鸭的病原的检测结果均呈阴性。

关键词：甲型鸭肝炎病毒；实时荧光定量RT-PCR；特异性；敏感性；重复性

Establishment of fluorescent quantitative PCR for duck hepatitis A virus 1 strain (DHAV-1)

Abstrac: In this study, a pair of upstream and downstream primers and corresponding TaqMan probes that can specifically amplify 140bp were designed based on the comparison of 84 VP1 gene nucleic acid sequences of DHAV-1, downloaded from NCBI database. By optimizing the concentration of primer, probe and annealing temperature, a real-time fluorescence quantitative RT-PCR detection method of duck hepatitis A virus 1 strain (DHAV-1) was established, and its specificity, sensitivity and repeatability were optimized. The results showed that this method exhibited a typical s-type amplification curve, which could specifically amplify the target fragment of about 140bp-sized. Its sensitivity reached 102 copies, and the coefficient of variation of intra- and inter-assay tests’ repetition was less than 5%. At the same time, the detection result of other seven ducks pathogens with this method were negative.

Key words: DHAV; qRT-PCR; Specificity; Sensitivity; Repeatability

目  录

摘要 1

关键词 1

Abstract 1

Key words 1

引言 1

1材料 2

1.1主要仪器 2

1.2主要试剂 2

1.3模板毒株 3

1.4载体与菌株 3

2方法 3

2.1引物与探针 3

2.2标准阳性质粒构建 4

2.3荧光定量PCR反应条件优化 8

2.4特异性、敏感性与重复性检验 8

3结果 8

3.1引物选择 8

3.2最佳引物、探针浓度及退火温度 9

3.3特异性检验结果 9

3.4敏感性检验结果 10

3.5重复性检验结果 11

4讨论 11

致谢 12

参考文献 13

Ⅰ 型1系鸭肝炎病毒（DHAV-1）实时荧光定量RT-PCR检测方法的建立

鸭病毒性肝炎是由鸭肝炎病毒（DHV）引起的一种具有高度传染性和致死性的病毒性疾病。病鸭早期阶段会出现精神沉郁、食欲不振、行动呆滞迟缓的症状，常蹲伏于地不愿四处走动觅食，半闭眼呈昏睡状[6]。雏鸭在发病后期死亡时常伴有较明显的神经症状，如双翅下垂、双腿痉挛抽搐，头颈向后背部扭曲呈现角弓反张的姿势，剖检可见明显的肝脏的水肿、出血和坏死[7]。幼龄的雏鸭对鸭肝炎病毒极易感，雏鸭的日龄越小则死亡率越高；后期随着雏鸭日龄的增加，将会逐渐获得免疫保护，死亡率明显下降。成年鸭几乎不发病，但大部分成年鸭呈隐性感染状态，可作为传染源，不断向外界排毒。因此，有必要做好针对DHV的预防和鉴别诊断工作，防止鸭病毒性肝炎的大规模爆发，减少经济损失，促进我国水禽养殖业发展。