甘蔗杆状病毒荧光定量PCR检测方法的建立及初步应用

为建立一种能够快速、灵敏、特异的检测甘蔗杆状病毒(sugarcane bacilliform virus,SCBV)的SYBR GreenⅠ荧光定量PCR方法,针对SCBV的基因序列,设计了特异性扩增引物,利用构建的标准品建立和优化针对SCBV的荧光定量PCR检测方法,并对该方法进行了特异性、稳定性、灵敏性等的测试,随后用于田间样品的检测。结果表明:将含有SCBV基因组序列的重组质粒进行梯度稀释制成标准品,利用标准品进行荧光定量PCR,获得标准曲线y=-3.4821x+37.264,相关系数r^2=0.9999,说明CT值与反应起始模板数量呈线性关系,可进行准确定量;组内和组间变异系数在0.19%~1.68%之间,表明检测方法重复性良好;建立的荧光定量PCR方法最低可检测到10个拷贝重组质粒/μL,是常规PCR检测灵敏度的100倍。使用建立的荧光定量PCR方法和常规PCR方法对采集的90份甘蔗叶片样品进行检测,常规PCR检出53份阳性样品,荧光定量PCR检出56份阳性样品,表明所建立的荧光定量PCR方法较常规PCR敏感性高,且准确性高。本研究建立的SCBV荧光定量PCR检测方法重复性好,灵敏度高,为构建甘蔗健康种苗体系提供了一种高效检测方法。

Establishment and Preliminary Application of Fluorescence Quantitative PCR for Detection of Sugarcane Bacilliform Virus

To establish a rapid,sensitive and specific SYBR GreenⅠfluorescence quantitative PCR method for the detection of sugarcane bacilliform virus(SCBV),according to SCBV gene sequence,the specific primers were designed,and the fluorescence quantitative PCR was established and optimized to detect SCBV by using the constructed plasmid that contained a SCBV genomic fragment.The specificity,stability and sensitivity of the method were tested,and then it was used for the detection of field samples.Results showed that the recombinant plasmid containing the SCBV genomic sequence was serially diluted and used as the amplification template of fluorescence quantitative PCR,according the result,a standard curve y=-3.4821x+37.264 was constructed,and the correlation coefficient r2=0.9999,which indicated that the CT value was linear with the copy number of reaction initiation templates.The intra-assay and inter-assay coefficient of variation was from 0.19%to 1.68%,which indicated that the detection method had good repeatability.The established fluorescence quantitative PCR method can detect 10 copies/μL of recombinant plasmid,which was 100 times more sensitive than conventional PCR.90 field samples were detected using both the fluorescence quantitative PCR method and conventional PCR method,56 positive samples were detected by fluorescence quantitative PCR,and 53 positive samples were detected by conventional PCR,which indicated that the established quantitative PCR method was sensitive and accuracy.This study provided an efficient detection method for constructing a healthy sugarcane seedling system.