中国动物传染病学报2011，Vol.19Issue(5)：8-14,7.
基因Ⅶ型新城疫病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立
DESIGN AND VALIDATION OF REAL TIME SYBR GREEN Ⅰ FLUORESCENCE QUANTITATIVE RT-PCR ASSAY FOR DETECTION OF GENOTYPE Ⅶ NEWCASTLE DISEASE VIRUS
摘要
Abstract
The real time SYBR Green Ⅰ fluorescence quantitative reverse transcription polymerase chain reaction（rRT-PCR） assay based on conserved region of matrix gene was developed for rapid detection of genotype Ⅶ Newcastle disease virus.The target sequence of M gene was cloned into the T-easy vector and a series of diluted recombinant plasmids were used to generate standard curve.The rRT-PCR assay had a detection limit of 100 copies of target DNA per-reaction and a correlation coefficient of 0.999.The assay showed good specificity and did not cross react with other viruses including genotype（Ⅱ,Ⅳ,Ⅸ） Newcastle disease virus,H5 Avian influenza virus,H9 Avian influenza virus and Infection bronchitis virus.The variation coefficients of intra assay were below 5%,which indicated good reproducibility.Forty experimentally infected samples were validated by the assay.The results showed that the detection rate was 95% compare with 70% by conventional RT-PCR.It showed better sensitivity and specificity of rRT-PCR than conventional RT-PCR.关键词
Ⅶ型新城疫病毒/SYBR/Green/Ⅰ/实时荧光定量RT-PCRKey words
Genotype Ⅶ Newcastle disease virus/SYBR Green Ⅰ/real time fluorescent quantitative RT-PCR分类
农业科技引用本文复制引用
孟春春,段云兵,仇旭升,金仕强,詹媛,张向乐,于圣青,丁铲..基因Ⅶ型新城疫病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立[J].中国动物传染病学报,2011,19(5):8-14,7.基金项目
农业公益性行业科研专项课题（201003012) （）
国家高科技研究发展计划863计划（2011AA10A200) （）
