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。
Establishment and application of a SYBR Green Ⅰ fluorescence quantitative PCR detection method for waterfowl parvoviruses
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.
waterfowl parvovirusesdetection methodSYBR Green Ⅰfluorescence quantitative PCR
NETL
NSTL
万方数据
