基于SYBR Green Ⅰ的桑树皱叶病毒qPCR检测方法的建立及应用  

Establishment and Application of SYBR GreenⅠBased qPCR Assay for the Detection of Mulberry Crinkle Leaf Virus

作　　者：孙鑫[1] 张健[1,2] 杨磊[1] 卢全有[1,2] Sun Xin;Zhang Jian;Yang Lei;Lu Quanyou(College of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212018,China;Sericul-tural Research Institute,Chinese Academy of Agricultural Sciences,Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture and Rural Affairs,Zhenjiang Jiangsu 212018,China)

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212018 [2]中国农业科学院蚕业研究所,农业农村部蚕桑遗传改良重点实验室,江苏镇江212018

出　　处：《蚕业科学》2020年第2期140-145,共6页ACTA SERICOLOGICA SINICA

基　　金：江苏省高校自然科学研究重大项目(18KJA210001)。

摘　　要：根据桑树皱叶病毒(mulberry crinkle leaf virus,MCLV)cp基因的核苷酸序列,设计了3对用于实时荧光定量PCR(quantitative real-time PCR,qPCR)的引物,以含有cp基因全长序列的重组质粒为模板,通过PCR和熔解曲线分析筛选出1对适合用于qPCR的特异性引物。以10倍梯度稀释的含有cp基因全长序列的重组质粒为模板,进行SYBR GreenⅠqPCR并制作标准曲线,建立了MCLV的qPCR检测方法。该方法对MCLV cp基因的检测灵敏度≥47.8个拷贝/μL。利用建立的qPCR方法对大田样品进行检测,结果显示15个样品都为MCLV感染阳性,其中病毒含量最高的样品的病毒拷贝数为5.26×10^4个拷贝/μL,最低的样品为8.36×10^2个拷贝/μL。MCLV qPCR检测体系的建立可以为培育健康桑树种苗、预防病毒病的发生提供精准保障技术,也为研究桑树不同生长时期或不同组织中病毒滴度变化奠定了基础。Three pairs of primers for quantitative real-time PCR(qPCR)were designed according to nucleotide sequence of cp gene from mulberry crinkle leaf virus(MCLV).Taking recombinant plasmid with full-length of cp gene sequence as template,a pair of specific primers for qPCR assay was screened out based on PCR detection and analysis on dissociation curve.Taking 10-fold serial diluted recombinant plasmid with full-length of cp gene sequence as template,standard curve of SYBR GreenⅠbased qPCR for the detection of MCLV was constructed.Its detection sensitivity of cp gene from MCLV was no less than 47.8 copy/μL.Detection results of field mulberry samples showed that all 15 samples were positive to MCLV,among which the highest viral copy number was 5.26×10^4 copy/μL,and the lowest was 8.36×10^2 copy/μL.Estab-lishment of SYBR Green I based qPCR for the detection of MCLV provides an accurate technology for breeding healthy mul-berry seedlings and controlling mulberry viral disease.It also facilitates the study on changes of viral titer in mulberry tree at different growth stages or in different tissues.

关 键 词：桑树皱叶病毒 SYBR GreenⅠ 实时荧光定量PCR(qPCR) 检测 灵敏度 

分 类 号：S888.71[农业科学—特种经济动物饲养]