土壤中南方根结线虫的实时荧光PCR检测和定量  

Detection and quantification of Meloidogyne incognita in soil sample using real-time PCR

作　　者：宋志强[1] 王暄[1] 林宇[1] 迟元凯[1] 鞠玉亮[1] 李红梅[1] 

机构地区：[1]南京农业大学植物保护学院,农作物生物灾害综合治理教育部重点实验室,南京210095

出　　处：《植物保护学报》2013年第3期255-260,共6页Journal of Plant Protection

基　　金：国家公益性行业(农业)科研专项(201103018);国家自然科学基金(30900939)

摘　　要：为了准确测定土壤中南方根结线虫Meloidogyne incognita的群体密度,根据序列特异性扩增区标记设计了南方根结线虫特异性引物和TaqMan探针,分别建立了卵和2龄幼虫的实时荧光PCR定量检测体系,并将该检测体系与显微镜计数法进行了比较。结果表明,引物Mi-F/R及探针Mi-probe对南方根结线虫具有高度特异性,建立的标准曲线循环阈值与卵和2龄幼虫数量的对数值之间具有良好的线性关系,相关系数分别为0.9771和0.9853,扩增效率均为90%,检测灵敏度为10-1个卵及10-3条2龄幼虫;对10个田间土壤样本的检测显示,实时荧光PCR定量检测与显微镜计数法测定的南方根结线虫卵和2龄幼虫数量呈显著正相关,且两种方法对2龄幼虫的测定结果无显著差异。：In order to accurately identify and quantify Meloidogyne incognita in soil, the real-time PCR assays for second-stage juveniles （J2） and eggs were developed. The specific primers Mi-F/R and TaqMan probe Mi-probe were designed according to the SCAR marker sequences of M.incognita. Correlation was analyzed between the nematode numbers obtained by real-time PCR estimation and traditional microscope counting. The results indicated that the primers and probe were highly specific to M.incognita. The established standard curves showed the strong linear relationship between the cycle threshold and the logarithmic values from the number of eggs and J2s. The correlation coefficient in the equation from eggs and J2s were 0.9771 and 0.9853, respectively. The amplification efficiencies of the real-time PCR assay for both were 90%, and the sensitivity were 10-1 egg and 10-3 J2, respectively. The detection of 10 field samples revealed there was a significantly positive correlation for the numbers of eggs and J2s of M.incognita in soil between the methods of the real-time PCR estimation and microscope counting, and there was no significant difference in estimating the number of J2s.

关 键 词：南方根结线虫 2龄幼虫 卵 实时荧光PCR TAQMAN探针 

分 类 号：S433[农业科学—农业昆虫与害虫防治]