摘要
为建立禽痘病毒(APV)快速灵敏的荧光定量PCR(qPCR)检测方法,本研究经PCR扩增鸡痘病毒(FPV,APV中的主要病毒)高度保守核心基因P4b,构建重组质粒标准品pMD19-FPV-P4b,经PCR与测序鉴定正确后作为模板,采用设计的qPCR通用型引物FPV-JC-F/R(根据分析比对不同种属27种APV A分支高度保守的核心基因P4b设计),利用方阵法优化各反应条件,初步建立检测APV的qPCR方法。将重组质粒pDM19-FPV-P4b 10倍倍比稀释后作为模板,利用优化的反应条件经qPCR扩增,以拷贝数的对数值为纵坐标,Ct值为横坐标绘制标准曲线,结果显示,标准曲线的R^(2)为0.9995,质粒标准品在3.53拷贝/μL~3.53×10^(8)拷贝/μL范围内与各自的Ct值呈良好的线性关系。采用建立的qPCR方法检测APV及禽类常见病毒,评估其特异性;采用该qPCR和常规PCR方法分别检测10倍倍比稀释的FPV弱毒疫苗(9.75×10^(6)TCID_(50)/mL~9.75×10^(-1)TCID_(50)/mL)及质粒标准品(3.53×107拷贝/μL~3.53拷贝/μL),评估该方法的敏感性;以不同浓度的质粒标准品作为模板,于同一时间和不同时间分别利用该qPCR方法检测,评估该方法的重复性。结果显示,除FPV与鸽痘病毒(PPV)检测结果为阳性外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸡传染性法氏囊病毒(IBDV)、鸡传染性喉气管炎病毒(ILTV)、禽腺病毒4型(FAdV-4)、火鸡疱疹病毒(HVT)等均为阴性结果,特异性强;该方法对FPV弱毒疫苗与质粒标准品的检测限分别为9.75 TCID_(50)/mL和3.53拷贝/μL,常规PCR对FPV弱毒疫苗和质粒标准品的检测限分别为975 TCID_(50)/mL和3.53×10^(2)拷贝/μL,组内组间重复性试验的变异系数均小于1.57%,重复性好。采用该方法和常规PCR方法同时检测186份临床疑似患鸡痘鸡的病料样品(鸡咽喉部痘疱结节、痘痂样品41份;肺、肝、肾脏样品各32份;咽拭子样品49份),结果显示,qPCR的阳性检出率为65.1%(121/186),常规PCR的阳性检出率为55.9%(104/186),二者检测结果的阳性符合率、阴性符合率与总符合率分别为100%、79.3%(65/82)与90.9%(169/186)。综上,本研究建立的检测APV的qPCR方法特异性强、敏感性高、重复性和稳定性好、适用范围更广,可以用于临床各种样品的快速检测,为APV的临床检测和流行病学调查提供了一种便捷的新的技术手段。
To develop a rapid and sensitive fluorescence quantitative PCR(qPCR)method for the detection of avian poxvirus(APV),the highly conserved core gene P4b of fowlpox virus(FPV,the main viruses in APV)was amplified by PCR in this study.A recombinant plasmid standard,pMD19-FPV-P4b,was constructed and verified through PCR and sequencing.Universal qPCR primers FPV-JC-F/R were designed based on the analysis and calignment of the P4b gene sequences from 27 APV A branch species.A preliminary qPCR pro-tocol for APV detection was established by using the primers above and optimizing the reaction conditions in an orthogonal-array format.The recombinant plasmid pMD19-FPV-P4b was subjected to 10-fold serial dilution and used as a template.Under the optimized reaction conditions,qPCR amplification was performed,and a standard curve was generated by plotting the logarithm of the copy number on the vertical axis against the Ct value on the horizontal axis.The results indicated that the coefficient of determination(R^(2))of the standard curve was 0.9995,demonstrating a strong linear correlation between the plasmid copy number and Ct values within the range of 3.53 copies/μL to 3.53×10^(8)copies/μL.The specificity of this qPCR assay was evaluted by testing APV and several common avian viruses.The sen-sitivity of this qPCR was assessed by testing the FPV attenuated vaccine(ranging from 9.75×10^(6)TCID_(50)/mL to 9.75×10^(-1)TCID_(50)/mL)and the plasmid standard subjected to 10-fold serial dilutions(from 3.53×107 copies/μL to 3.53×10^(-1)copies/μL).The repeatability of this qPCR was analyzed by testing different copy numbers of the plasmid standard(ranging from 3.53×107 copies/μL to 3.53×104 copies/μL)simultaneously or at different time points.The results showed that,except for FPV and pigeonpox virus(PPV),all other tested avian viruses-including Newcastle disease virus(NDV),H9 subtype avian influenza virus(H9 AIV),infectious bursal disease virus(IBDV),infectious laryngotracheitis virus(ILTV),avian adenovirus 4(FAdV-4),and turkey herpesvirus(HVT)-yielded negative results,indicating high specificity of the method.The detection limits of qPCR were 9.75 TCID_(50)/mL and 3.53 copies/μL for the FPV attenuated vaccine and plasmid standard,respectively.In comparison,those of the conventional PCR were 975TCID_(50)/mL and 3.53×10^(2)copies/μL,respectively.The intra-and inter-assay coefficients of variation were both below 1.57%,indicating excellent repeatability.A total of 186 clinical samples suspected of avian pox(including 41 throat papules and scab samples,32 each of lung,liver,and kidney samples,and 49 throat swab samples)were tested using both qPCR and conventional PCR methods.The positive detection rate was 65.1%(121/186)for qPCR,while that was 55.9%(104/186)for conventional PCR.The positive agreement rate,negative agreement rate,and overall agreement rate were 100%,79.3%(65/82),and 90.9%(169/186)between the set wo methods,In conclusion,it is demonstrated that the qPCR assay developed in this study for APV detection is strong specificity,high sensitivity,good repeatability,and stability,along with broad applicability.It is suitable for the rapid detection of various clinical samples and provides a convenient and effective technical tool for the clinical diagnosis and epidemiological investigation of APV.
作者
叶伟成
陈柳
倪征
云涛
华炯钢
霍苏馨
付媛
张存
朱寅初
YEWei-cheng;CHEN Liu;NI Zheng;YUN Tao;HUA Jiong-gang;HUO Su-xin;FU Yuan;ZHANG Cun;ZHU Yin-chu(Zhejiang Key Laboratory of Livestock and Poultry Biotech Breeding,Key Laboratory of Livestock and Poultry Resources(Poultry)Evaluation and Utilization,Ministry of Agriculture and Rural Affairs,Zhejiang Engineering Research Center for Poultry Breeding Industry and Green Farming Technology,Institute of Animal Husbandry and Veterinary Sciences,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)
出处
《中国预防兽医学报》
北大核心
2025年第8期813-819,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
浙江省“尖兵”“领雁”研发攻关计划(2023C02022)
浙江省农业重大技术协同推广项目(2023ZDXT15)
浙江省“三农九方”科技协作计划项目(2023SNJF042)。
