贵州农业科学
貴州農業科學
귀주농업과학
GUIZHOU AGRICULTURAL SCIENCES
2014年
6期
104-108
,共5页
鲜思美%张素辉%杨钰%邓诗%吴健%刘嫒%刘宗胜
鮮思美%張素輝%楊鈺%鄧詩%吳健%劉嬡%劉宗勝
선사미%장소휘%양옥%산시%오건%류애%류종성
羊口疮病毒%实时荧光定量 PCR%检测方法
羊口瘡病毒%實時熒光定量 PCR%檢測方法
양구창병독%실시형광정량 PCR%검측방법
ORFV%real-time fluorescence quantification PCR%detection method
为羊口疮（Orf）的分子流行病学调查、早期快速诊断及细胞培养物的检测等提供参考，根据GenBank 发表的羊口疮病毒（OrfV）B2L 基因序列设计合成1对引物，以 pMD18-T-B2L 重组质粒为阳性标准品，建立了检测 OrfV 核酸的 SYBR Green Ⅰ实时荧光定量 PCR 方法，并进行了临床应用。结果表明：建立的 SYBR Green Ⅰ实时荧光定量 PCR 的线性关系良好，标准曲线的相关系数达到-1；最低检测限为1．0×103 copies／μL，比常规 PCR 方法高100倍，与山羊痘和口蹄疫疫苗毒均不发生交叉反应；组内变异系数为1．061％～2．873％，组间变异系数为0．397％～3．829％。用该方法检测5例 OrfV 感染临床病例和8份不同代次的 OrfV 细胞培养物，阳性率均为100％。SYBR Green Ⅰ实时荧光定量 PCR 方法灵敏度高、特异性强、重复性好，可以用于 OrfV 的病原检测及定量分析。
為羊口瘡（Orf）的分子流行病學調查、早期快速診斷及細胞培養物的檢測等提供參攷，根據GenBank 髮錶的羊口瘡病毒（OrfV）B2L 基因序列設計閤成1對引物，以 pMD18-T-B2L 重組質粒為暘性標準品，建立瞭檢測 OrfV 覈痠的 SYBR Green Ⅰ實時熒光定量 PCR 方法，併進行瞭臨床應用。結果錶明：建立的 SYBR Green Ⅰ實時熒光定量 PCR 的線性關繫良好，標準麯線的相關繫數達到-1；最低檢測限為1．0×103 copies／μL，比常規 PCR 方法高100倍，與山羊痘和口蹄疫疫苗毒均不髮生交扠反應；組內變異繫數為1．061％～2．873％，組間變異繫數為0．397％～3．829％。用該方法檢測5例 OrfV 感染臨床病例和8份不同代次的 OrfV 細胞培養物，暘性率均為100％。SYBR Green Ⅰ實時熒光定量 PCR 方法靈敏度高、特異性彊、重複性好，可以用于 OrfV 的病原檢測及定量分析。
위양구창（Orf）적분자류행병학조사、조기쾌속진단급세포배양물적검측등제공삼고，근거GenBank 발표적양구창병독（OrfV）B2L 기인서렬설계합성1대인물，이 pMD18-T-B2L 중조질립위양성표준품，건립료검측 OrfV 핵산적 SYBR Green Ⅰ실시형광정량 PCR 방법，병진행료림상응용。결과표명：건립적 SYBR Green Ⅰ실시형광정량 PCR 적선성관계량호，표준곡선적상관계수체도-1；최저검측한위1．0×103 copies／μL，비상규 PCR 방법고100배，여산양두화구제역역묘독균불발생교차반응；조내변이계수위1．061％～2．873％，조간변이계수위0．397％～3．829％。용해방법검측5례 OrfV 감염림상병례화8빈불동대차적 OrfV 세포배양물，양성솔균위100％。SYBR Green Ⅰ실시형광정량 PCR 방법령민도고、특이성강、중복성호，가이용우 OrfV 적병원검측급정량분석。
The detection method of SYBR Green I real-time fluorescence quantification PCR for ORFV nucleic acid was established based on the positive standard of pMD18-T-B2L recombinant plasmid according to OPFV B2L gene sequence recommended by GenBank and the detection method was used in clinical application to provide a reference for ORFV molecular epidemiological investigation;early rapid diagnosis and detection of cell culture.The results showed that the established SYBR Green I real-time fluorescence quantification PCR has a good linear relation and the correlation coefficient of the standard curve was up to -1,and the lowest detection limit was 1.0 × 103 copies/μL,100 times more than the conventional PCR. Moreover,there was no cross reaction with GPV and FMDV, and the variable coefficient of intra-group and inter-group was 1.061%~2.873% and 0.397%~3.829% respectively.The positive rate of 5 ORFV clinical cases and 8 ORFV cell cultures with different generation detected by the established SYBR Green I real-time fluorescence quantification PCR was 100% all.The established SYBR Green I real-time fluorescence quantification PCR with high sensitivity, strong specificity and good repeatability can be used in detection and quantitative analysis of ORFV pathogen.
