摘要
在我国,肠道病毒71型(enterovirus 71,EV71)C4型是引起手足口病的主要流行基因型。为建立EV71C4型TaqMan荧光定量PCR检测方法,在C4型EV71VP1基因的高保守区,设计合成引物和TaqMan探针,将包含此目的区段的基因片段克隆到pcDNA3.1载体中,通过体外转录获得标准品,并以梯度稀释的标准品为模板建立工作曲线,进而在优化反应条件的基础上建立TaqMan荧光实时定量PCR检测方法。实验中,所设计引物、探针的高度保守性保证了C4型EV71的高效扩增。经反应条件优化,引物和探针的最佳工作浓度分别为300 nmol/L和200 nmol/L,在1×10~301×10~3拷贝数检测范围内具有良好的线性关系(R^2=1),灵敏度可达到10~2 copies/μL。通过对该方法进行检验发现,批间和组间重复实验的变异系数均小于0.5%,且该方法对柯萨奇A16(coxsackievirus A16,CA16)柯萨奇B1(coxsackievirus B1,CB1)人轮状病毒(human rotavirus,HRV)单纯疱疹病毒2型(herpes Simplex virus type 2,HSV-2)均无交叉反应,对6份EV71阳性样本检出率为100%。以上数据表明,文中建立的TaqMan荧光定量PCR方法可为我国主要流行C4型EV71感染的快速诊断及疾病监控提供有效途径。
C4 is the mainly prevalent genotype of enterovirus 71 （EV71） in China,which can cause hand, foot and mouth disease. To develop a quantitative TaqMan PCR assay for the detection of EV71, a set of primers and TaqMan probe were designed based on the highly conserved region in VP1 gene of C4. The target gene was obtained and cloned into the vector pcDNA3.1＋, and the transcribed RNA in vitro was diluted to get the standard RNA for setting up the standard curve. After the PCR conditions were optimized, the set of primers and probe were proved to be used for detecting the EV71 C4 subtype efficiently, and the optimal concentrations of the primers and probe were 300 nmol/L and 200 nmol/L, respectively. When viral load ranged from 1×103 to 1×109 copies, the R2 value was 1. The sensitivity of PCR reached 102 copies/μL. The inter- and intra-assay coefficient of variation was less than 0.5%, and no cross-reaction was found with coxsackievirus A16 （CA16）, coxsackievirus B1 （CB1）, human rotavirus （HRV） or herpes simplex virus type 2 （HSV-2）. What＇s more, six samples with EV71 were effectively identified to be positive. The results showed that the established quantitative PCR assay could provide a more rapid and useful way to diagnose and con- trol hand, foot and mouth disease in China.
出处
《生命科学研究》
CAS
CSCD
2016年第3期189-195,共7页
Life Science Research
基金
国家科技支撑计划(2014BAI01B01)
关键词
肠道病毒71型
VP1基因
高保守区
TAQMAN探针
荧光定量PCR
检测
C4 is the mainly prevalent genotype of enterovirus 71 （EV71） in China,which can cause hand, foot and mouth disease. To develop a quantitative TaqMan PCR assay for the detection of EV71, a set of primers and TaqMan probe were designed based on the highly conserved region in VP1 gene of C4. The target gene was obtained and cloned into the vector pcDNA3.1＋, and the transcribed RNA in vitro was diluted to get the standard RNA for setting up the standard curve. After the PCR conditions were optimized, the set of primers and probe were proved to be used for detecting the EV71 C4 subtype efficiently, and the optimal concentrations of the primers and probe were 300 nmol/L and 200 nmol/L, respectively. When viral load ranged from 1×103 to 1×109 copies, the R2 value was 1. The sensitivity of PCR reached 102 copies/μL. The inter- and intra-assay coefficient of variation was less than 0.5%, and no cross-reaction was found with coxsackievirus A16 （CA16）, coxsackievirus B1 （CB1）, human rotavirus （HRV） or herpes simplex virus type 2
