检测猪粪便中基因4型戊型肝炎病毒的实时荧光定量RT-PCR方法的建立  

Establishment of a Fluorescent Real-time Quantitative RT-PCR Assay for Detection of Genotype 4 Hepatitis E Virus in Swine Stools

作　　者：贾鹏[1,2] 金宁一[1] 李霄[1] 朱光泽[3] 刘妍[1,2] 高鹏[1,2] 徐晓红[1,2] 杨恩成[1,2] 孟日增 阚式绂[1,2] 

机构地区：[1]军事医学科学院全军基因工程重点实验室,长春130062 [2]吉林大学,长春130062 [3]长春中医药大学,长春130021 [4]吉林省进出口检验检疫局,长春130062

出　　处：《病毒学报》2010年第1期33-39,共7页Chinese Journal of Virology

基　　金：国家自然科学基金(30771609)

摘　　要：针对基因4型戊型肝炎病毒(Hepatitis E virus,HEV)基因组ORF3设计特异性引物和探针,建立了一套TaqMan实时荧光定量RT-PCR方法。评价了该方法的准确性和灵敏性,同时与常规RT-PCR和巢式RT-PCR方法进行了对比分析。结果表明,生成的标准曲线显示各浓度范围内具有良好的线性关系,相关系数(r2)为0.994,斜率为-3.312,PCR效率为100%。组内和组间重复性试验相同稀释度标准质粒Ct值之间差异均不显著,并且能够准确的从HEV阳性猪粪便样品中检测到基因4型HEV RNA,具有较好准确性。可检测到戊型肝炎病毒数量为(1.7×101)copies,比普通RT-PCR的灵敏度高100倍,比Nested RT-PCR高10倍。The primers and probes for the Real-time RT-PCR were designed based on the multiple sequence （swine and humans HEV strains） alignments of the ORF3 region of genotype 4 HEV. A rapid, sensitive and stable TaqMan Real-time RT-PCR assay was established, and its specificity and sensitivity were assessed, and comparison of the Real-time RT-PCR with conventional and nested RT-PCR was performed. The results found that the crossing points showed linearly proportional to the logarithm of the input copy number. The correlation coefficient （R^2） and the slope value of the standard curves with plasmid DNA were 0. 994 and -3. 312, respectively. The efficiency （E） of the PCR was 100%. Coefficients of variation values of the different diluted plasmid DNA were low in the same or different repeated experimental group. In addition, the assay was able to correctly detect genotype 4 HEV RNA from swine fecal samples. The sensitivity of established assay was 100-fold higher than that of conventional RT-PCR and 10-fold higher than nested RT-PCR.

关 键 词：戊型肝炎病毒 RT-PCR 基因4型 

分 类 号：R373.9[医药卫生—病原生物学]