水禽细小病毒SYBR Green Ⅰ荧光定量PCR检测方法的建立与应用
Establishment and application of a SYBR Green Ⅰ fluorescence quantitative PCR detection method for waterfowl parvoviruses
汪宏才 1商雨 1马瑶 2曾哲 1张蓉蓉 1姚伦 1罗玲 1李丽 2温国元 1罗青平1
作者信息
- 1. 湖北省农业科学院畜牧兽医研究所,武汉 430064;湖北省农业科学院农业农村部畜禽细菌病防治制剂创制重点实验室,武汉 430064;湖北省农业科学院畜禽病原微生物学湖北重点实验室,武汉 430064;湖北洪山实验室,武汉 430070
- 2. 湖北省农业科学院畜牧兽医研究所,武汉 430064
- 折叠
摘要
为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法.该方法的扩增效率(E)为90.0％,相关系数(R2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15％～0.23％,批间变异系数为0.09％～0.28％.结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强.临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4％,灵敏度是普通PCR的1 000倍.SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测.
Abstract
In order to establish a rapid detection method for waterfowl parvoviruses(WPV),specific primers were designed within the conserved SF3 region of the NS gene of waterfowl parvoviruses based on sequence alignment results,and a SYBR Green Ⅰ fluorescence quantitative PCR universal detection method was established.The amplification efficiency(E)of this method was 90.0％,the correla-tion coefficient(R2)was 0.99,and the standard curve equation was y=-3.607x+38.77;except for WPV with an S-shaped amplification curve,the newcastle disease virus(NDV),H9 subtype avian influenza virus(H9 AIV),duck tembusu virus(DTMUV),duck hepa-titis A virus(DHAV),duck enteritis virus(DEV),and duck reovirus(DRV)samples did not show an S-shaped positive amplifica-tion curve;the coefficient of variation(CV)within a batch was 0.15％to 0.23％,and the coefficient of variation between batches was 0.09％to 0.28％.The results indicated that the SYBR Green Ⅰ fluorescence quantitative PCR detection method had good repeatability,high sensitivity,and strong specificity.The clinical sample testing results showed that the coincidence rate between SYBR Green Ⅰ flu-orescence quantitative PCR and conventional PCR was 98.4％,and the sensitivity was 1 000 times higher than that of conventional PCR.The SYBR Green Ⅰ fluorescence quantitative PCR detection method could not only qualitatively detect WPV,but also quantita-tively detect it.It could be used for WPV purification detection in duck and goose breeding farms,as well as for rapid detection of WPV in large clinical samples.
关键词
水禽细小病毒/检测方法/SYBR/Green/Ⅰ/荧光定量PCRKey words
waterfowl parvoviruses/detection method/SYBR Green Ⅰ/fluorescence quantitative PCR引用本文复制引用
基金项目
湖北省产业链项目(2022HSZD005)
湖北省农业科技创新中心项目(2021-620-000-001-17)
出版年
2024
NETL
NSTL
万方数据