摘要
【目的】鸽腺病毒感染在全球鸽群中广泛分布,可引起多种临床症状,如呕吐、腹泻、肝脏损伤等,严重影响鸽的健康与养殖效益。建立一种快速高效检测鸽腺病毒Ⅰ型(PiAdV-Ⅰ)TaqMan探针实时荧光定量PCR方法,为鸽腺病毒Ⅰ型临床检测及流行病学调查提供技术平台。【方法】依据GenBank公布鸽腺病毒Ⅰ型Hexon基因保守区设计引物探针并构建重组质粒pCE2-TA-PiAdV-Ⅰ;优化反应体系和程序,绘制标准曲线、特异性、敏感性和重复性评价,建立鸽腺病毒Ⅰ型TaqMan探针实时荧光定量PCR检测方法;扩增测序阳性临床样品的Hexon基因,使用MEGA5.0进行进化树构建及同源性分析。【结果】该TaqMan探针实时荧光定量PCR方法标准曲线y=-3.1081x+37.374,线性相关系数R2为0.9977,线性关系良好;与鸽疱疹病毒、鸽圆环病毒等鸽子其他常见病毒无交叉反应,具有良好的特异性;最低检测拷贝数为14.6 copies/μL,敏感性是常规PCR方法的10倍,具有高敏感性;组内和组间变异系数均低于1.1%,重复性好。通过检测112份鸽子病料样品,该TaqMan探针实时荧光定量PCR方法检测阳性率为69.64%(78/112),并且高于常规PCR方法阳性检出率65.18%(73/112)。此外,建立的TaqMan探针实时荧光定量PCR方法与常规PCR方法的阳性符合率为100%,总体符合率为95.54%。3条测序毒株与Ⅰ型鸽腺病毒参考毒株同源性为99.47%~99.71%,遗传关系高度接近,表明可能由同一个原始毒株进化而来。【结论】研究建立的鸽腺病毒Ⅰ型TaqMan探针实时荧光定量PCR检测方法线性关系良好、特异性强、敏感性高及重复性好,可快速高效检测出临床样品中的PiAdV-Ⅰ。此外,所扩增序列能够用于分析临床毒株遗传进化特征,有助于快速准确诊断鸽腺病毒感染及疫苗研发提供参考依据。
[Objective]Pigeon adenovirus infection is widely distributed in pigeon populations around the world,which can cause a variety of clinical symptoms,such as vomiting,diarrhea,liver damage,etc.,seriously affecting the health of pigeons and breeding benefits.This study aims to establish a rapid and efficient TaqMan probe quantitative PCR method for the detection of Pigeon Adenovirus type Ⅰ(PiAdV-Ⅰ),thus providing a technical platform for clinical detection and epidemiological investigation of pigeon adenovirus type Ⅰ.[Method]According to the conserved region of Hexon gene of pigeon adenovirus type Ⅰ published in GenBank,the primers and probes were designed and the recombinant plasmid pCE2-TA-PiAdV-Ⅰ was constructed.Based on the optimized reaction system and procedure,the standard curve was drawn,the specificity,sensitivity and repeatability were evaluated,and the pigeon adenovirus type Ⅰ TaqMan probe quantitative PCR assay was established.Hexon gene was amplified and sequenced from PiAdV-Ⅰ positive clinical samples.Phylogenetic tree and homology analysis were performed using MEGA5.0 software.[Result]The standard curve of the TaqMan probe quantitative PCR method was y=-3.108 1x+37.374,and the linear correlation coefficient R2 was 0.997 7,indicating a good linear relationship.There is no cross-reaction with other common pigeon viruses,such as pigeon herpesvirus and pigeon circovirus,demonstrating good specificity.The minimum detection copy number was 14.6 copies/μL,which was 10 times more sensitive than the conventional PCR method.The intra-and inter-group coefficients of variation were less than 1.1%,indicating good repeatability.Through the detection of 112 pigeon samples,the positive rate of the TaqMan probe quantitative PCR method was 69.64%(78/112) higher than that of the conventional PCR method 65.18%(73/112).In addition,the positive coincidence rate between the established TaqMan probe quantitative PCR method and the conventional PCR method was 100%,and the overall coincidence rate was 95.54%.The homology between the three sequenced strains and the reference strain of pigeon adenovirus type Ⅰ was 99.47%-99.71%,and the genetic relationship was highly close,indicating that the three sequenced strains may have evolved from the same original strain.[Conclusion]The established TaqMan probe quantitative PCR assay for the detection of pigeon adenovirus type Ⅰ has good linear relationship,strong specificity,high sensitivity and good repeatability,which can quickly and efficiently detect PiAdV-Ⅰ in clinical samples.In addition,the amplified sequences can be used to analyze the genetic evolution characteristics of clinical strains,which is helpful for rapid and accurate diagnosis of pigeon adenovirus infection and provides reference for vaccine development.
作者
安乐乐
刘倩芸
蓝秋菊
罗迅
赵永清
AN Lele;LIU Qianyun;LAN Qiuju;LUO Xun;ZHAO Yongqing(Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu university,Lanzhou 730100,China)
出处
《江西农业大学学报》
北大核心
2025年第1期167-177,共11页
Acta Agriculturae Universitatis Jiangxiensis
基金
甘肃省自然科学基金项目(21JR1RA221)。
