[1]张小敏,周斌,何丹妮,等.实时荧光定量PCR检测猪源Mx1方法的建立及应用[J].南京农业大学学报,2013,36(1):92-96.[doi:10.7685/j.issn.1000-2030.2013.01.016]
 ZHANG Xiaomin,ZHOU Bin,HE Danni,et al.Establishment and application of the SYBR GreenⅠreal-time PCR assay for detection of porcine antiviral protein Mx1 gene[J].Journal of Nanjing Agricultural University,2013,36(1):92-96.[doi:10.7685/j.issn.1000-2030.2013.01.016]
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实时荧光定量PCR检测猪源Mx1方法的建立及应用()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
36卷
期数:
2013年1期
页码:
92-96
栏目:
出版日期:
2013-01-24

文章信息/Info

Title:
Establishment and application of the SYBR GreenⅠreal-time PCR assay for detection of porcine antiviral protein Mx1 gene
作者:
张小敏 周斌 何丹妮 庞然 赵津 陈溥言
南京农业大学农业部动物疫病诊断与免疫重点开放实验室, 江苏 南京 210095
Author(s):
ZHANG Xiaomin ZHOU Bin HE Danni PANG Ran ZHAO Jin CHEN Puyan
Key Laboratory of Animal Diseases Diagnosis and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
关键词:
猪源Mx1实时荧光定量PCR标准曲线
Keywords:
porcine Mx1 gene(poMx1)real-time PCR quantitative assaystandard curve
分类号:
S854.4+3
DOI:
10.7685/j.issn.1000-2030.2013.01.016
摘要:
根据GenBank中的猪源抗病毒蛋白Mx1基因(poMx1)序列设计1对特异性引物,从Mx1基因全长片段中扩增和克隆了特异性的115 bp核苷酸片段。测序鉴定正确后将重组质粒进行定量并10倍倍比稀释作为扩增模板,通过SYBR GreenⅠ实时荧光定量PCR方法,建立Mx1的标准曲线及回归方程。结果显示:该标准曲线的回归方程为Y=-2.986 3 lgX+38.239 3(R2=0.998 5),灵敏度为1×102 μL-1;应用该方法对猪瘟兔化弱毒苗接种猪肾细胞(PK-15)后产生的poMx1 mRNA进行定量分析,发现病毒感染细胞4 h后,poMx1的表达量最高(P<0.01),然后随着时间的增加而缓慢减少。结论:建立的SYBR GreenⅠ实时荧光定量PCR方法可以检测poMx1 mRNA的表达水平。
Abstract:
On the basis of the nucleotide sequences of porcine Mx1 gene(poMx1)available in GenBank,a pair of primers was designed and synthesized to amplify a specific 115 bp nucleotide fragment from Mx1 full-length gene.Then,the SYBR Green Ⅰ real-time PCR assay was established based on 10-fold dilution recombinant plasmid as the amplification template after it was cloned and sequenced.The results showed that the standard curve regression equation was Y=-2.986 3 lgX+38.239 3(R2=0.998 5),and that the standard curve was of high linearity,specificity,sensitivity and reproducibility.The production of poMx1 mRNA from virus-infected cells was quantitatively analyzed after swine kidney cells(PK-15)was stimulated by classical swine fever virus(HCLV strain).The data showed the production of poMx1 mRNA was much more after 4 h(P<0.01),and then slowly reduced as time grew.So the results showed that this PCR assay could be applied to detect the expression level of poMx1 mRNA.

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备注/Memo

备注/Memo:
收稿日期:2012-5-18。
作者简介:张小敏,硕士研究生。
通讯作者:周斌,博士,副教授,主要从事动物病毒分子免疫学研究,E-mail:zhoubin@njau.edu.cn。
更新日期/Last Update: 1900-01-01