Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita
Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita
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DOI：
10.1111/j.1747-0765.2007.00212.x
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发表时间：
2008-02
影响因子：
2
通讯作者：
K. Toyota;Tomoaki Shirakashi;E. Sato;S. Wada;Y. Min
中科院分区：
文献类型：
--
作者：
K. Toyota;Tomoaki Shirakashi;E. Sato;S. Wada;Y. Min
摘要设计引物PCN 280 f和NEPCN 398 r，应用实时荧光定量PCR技术对马铃薯胞囊线虫Globodera rostochiensis进行检测。分别以1、5、50、125和250头二龄幼鱼（J2）为试材，进行了室内饲养试验。rostochiensis与各阶段的蠕虫状秀丽隐杆线虫混合，得到总共500个个体，并从线虫混合物中提取DNA。循环阈值与G数呈显著正相关（r2 = 0.9355，P < 0.001）。rostochiensis补充说。从人工感染G.用提取的线虫DNA为模板进行实时荧光定量PCR检测，结果表明，线虫的数量与线虫的数量呈极显著正相关。从real-time PCR方法和形态学鉴定方法对Rostochiensis J2进行了初步鉴定。实时PCR灵敏地检测到单个G。rostochiensis J2的1,000个个体的自由生活的线虫。同样，设计了用于检测南方根结线虫的实时荧光PCR引物RKNf和RKNr。本研究表明，马铃薯孢囊线虫和根结线虫的实时荧光PCR检测方法为这些蠕虫状害虫的快速定量提供了一种灵敏可靠的手段。
Abstract The primers PCN280f and NEPCN398r were designed for the quantitative detection of the potato-cyst nematode Globodera rostochiensis using real-time polymerase chain reaction (PCR). One, five, 50, 125 and 250 individuals of the second-stage juveniles (J2) of G. rostochiensis were mixed with various stages of vermiform Caenorhabditis elegans to make a total of 500 individuals and DNA was extracted from the nematode mixture. There was a significant correlation (r 2 = 0.9355, P < 0.001) between the threshold cycle values and the number of G. rostochiensis added. When nematodes were extracted from soils artificially infested with G. rostochiensis to various degrees and real-time PCR was conducted using DNA templates from the nematodes extracted, there was a highly significant correlation in the numbers of G. rostochiensis J2 from the real-time PCR method and morphological identification. Real-time PCR sensitively detected a single G. rostochiensis J2 out of 1,000 individuals of free-living nematodes. Similarly, real-time PCR primers RKNf and RKNr were designed for the detection of the root-knot nematode Meloidogyne incognita. This study demonstrated that the real-time PCR assay for the potato-cyst nematode and the root-knot nematode provides a sensitive and reliable means for the rapid quantification of these vermiform pests.