禽腺病毒血清4型纳米PCR检测试剂盒的研制及应用

为研制一种高效、快捷、灵敏的禽腺病毒血清4型（FAdV-4）检测试剂盒，本研究根据FAdV-4的保守序列设计了一对引物及探针，并在下游引物标记FITC，探针标记Biotin，应用纳米PCR技术结合横向流动试纸条技术（LFD），优化反应及杂交条件后建立了检测FAdV-4的纳米PCR-LFD方法，并组装了FAdV-4纳米PCR检测试剂盒。同时对试剂盒进行了特异性、敏感性、批内批间可重复性、稳定性、保存期评估等试验及临床样品检测。特异性试验表明，此试剂盒能够特异性检测出FAdV-4，而对于禽的其他主要病毒性及细菌性病原如鸡传染性喉气管炎病毒（ILTV）、鸡传染性支气管炎病毒（IBV）、鸡传染性法氏囊病病毒（IBDV）、鸡马立克病病毒（MDV）、鸡减蛋综合征病毒（EDSV）、鸭瘟病毒（DPV）、新城疫病毒（NDV）、禽流感病毒H9亚型（AIV（H9））、禽呼肠孤病毒（ARV）、FAdV标准株（FAdV-1、FAdV-2、FAdV-8a、FAdV-8b和FAdV-11）、鸡大肠杆菌（E.coli）、金黄色葡萄球菌（SA）、鸡白痢沙门氏菌（SP）、鸡毒支原体（MG）的检测结果均为阴性。敏感性试验表明，此试剂盒的敏感性比常规PCR方法敏感10倍，最低核酸拷贝数检出量可以达到56.0拷贝/μL。试剂盒的批内、批间检测结果具有良好的一致性和稳定性。稳定性试验结果说明试剂盒至少可以耐受20次的反复冻融，依然有很好的检测效果。保存期试验证实，试剂盒在4 ℃条件下至少可以保存3个月，在-20 ℃条件下至少可保存12个月。此试剂盒实现了对FAdV-4检测结果的可视化，简化了操作流程，提高了检测效率。对天津及周边地区临床送检的117份样品检测结果显示，FAdV-4阳性率为17.95%（21/117）。本试验研制的试剂盒可用于FAdV-4的临床诊断和分子流行病学调查，对养禽场FAdV-4感染的早期检测与制定综合防控措施提供了重要的技术支撑。

Development and Application of Nanoparticle-assisted PCR Detection Kit for Fowl Aviadenovirus Serotype 4

This experiment was aimed to establish an efficient,fast and sensitive kit for fowl aviadenovirus serotype 4 (FAdV-4),the primers and probe were designed from penton gene of FAdV-4,and FITC was labeled in the downstream primer,Biotin was labeled in the probe.The nano PCR-LFD method for detection of FAdV-4 was established by using nano PCR technology and LFD technology,and the reaction and hybridization conditions were optimized.At the same time,the specificity,sensitivity,intra- and inter-batch repeatability,stability,shelf-life evaluation and clinical samples of the kit were tested.The specificity test showed that the kit could detect FAdV-4 specifically,while for other major viral and bacterial pathogens of poultry,such as infectious laryngotracheitis virus (ILTV),infectious bronchitis virus (IBV),infectious bursal disease virus (IBDV),chicken Marek's disease virus (MDV),egg drop syndrome virus (EDSV),duck plague virus (DPV),Newcastle disease virus (NDV),avian influenza virus H9 subtype (AIV (H9)),avian reovirus (ARV),FAdV standard strain (FAdV-1,FAdV-2,FAdV-8a,FAdV-8b and FAdV-11),E.coli,Staphylococcus aureus (SA),Salmonella Pullorum (SP) and Mycoplasma gallisepticum (MG) were all negative.The sensitivity detections suggested that the kit was so sensitive that it could detect the 56.0 copies/μL level and 10 folds higher than conventional PCR.The results of intra- and inter-repeatability test showed that it had good consistency and stability.The stability test results showed that the kit could withstand at least 20 times of repeated freezing and thawing,and still had a good detection effect.The shelf life test showed that the kit could be stored for at least 3 months at 4 ℃ and 12 months at -20 ℃.The kit realized the visualization of the detection results of FAdV-4,simplified the operation process and improved the detection efficiency.The positive rate of FAdV-4 was 17.95% (21/117).It could be used in the clinical diagnosis and molecular epidemiological investigation of FAdV-4,which provided an important technical support for the early detection of FAdV-4 infection in poultry farms and the formulation of comprehensive prevention and control measures.