Multiplex PCR for the Simultaneous Identification and Detection of Meloidogyne incognita, M. enterolobii, and M. javanica Using DNA Extracted Directly from Individual Galls
Multiplex PCR for the Simultaneous Identification and Detection of Meloidogyne incognita, M. enterolobii, and M. javanica Using DNA Extracted Directly from Individual Galls
复制标题
使用直接从个体虫瘿中提取的 DNA 进行多重 PCR 同时鉴定和检测南方根结线虫、肠杆菌和爪哇根结线虫
DOI：
10.1094/phyto-04-11-0095
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发表时间：
2011-11-01
期刊：
影响因子：
3.2
通讯作者：
Liao, J. L.
中科院分区：
文献类型：
--
作者：
Hu, M. X.;Zhuo, K.;Liao, J. L.
南方根结线虫（Meloidogyne incognita）、南方根结线虫M. enterolobii和M.爪哇线虫（javanica）是我国南方分布最广的一种根结线虫，危害着许多重要的经济作物、观赏植物和果树。本研究设计了1对根结线虫通用引物和3对种特异性引物，成功地应用于根结线虫的快速检测和鉴定。incognita，M。enterolobii和M.通过多重聚合酶链反应（PCR）使用从个体虫瘿中提取的DNA对javanica进行鉴定。所有M. incognita，M. enterolobii和M. javanica分离物分别产生约500和1，000、500和200以及500和700 bp的两个片段。500-bp片段是使用通用引物产生的rDNA 28 S D2/D3的内部阳性对照片段。其他根结线虫属包括在本研究中的片段仅产生一个大小约为500 bp的片段。使用这种方法，M。incognita，M. enterolobii和M.爪哇根结线虫的鉴定和检测使用DNA提取直接从个别的虫瘿含有根结线虫属。在它们生命周期的不同阶段。PCR阳性率随线虫的发育而增加，一般在第二阶段幼虫后期后较易检测。该协议适用于虫瘿从自然感染的根，结果被发现是快速，灵敏，稳健，准确。本研究首次为M. incognita，M. enterolobii和M. javanica使用DNA提取直接从个别的虫瘿使用一步多重PCR技术。
Meloidogyne incognita, M. enterolobii, and M. javanica are the most widespread species of root-knot nematodes in South China, affecting many economically important crops, ornamental plants, and fruit trees. In this study, one pair of Meloidogyne universal primers was designed and three pairs of species-specific primers were employed successfully to rapidly detect and identify M. incognita, M. enterolobii, and M. javanica by multiplex polymerase chain reaction (PCR) using DNA extracted from individual galls. Multiplex PCR from all M. incognita, M. enterolobii, and M. javanica isolates generated two fragments of approximate to 500 and 1,000, 500 and 200, and 500 and 700 bp, respectively. The 500-bp fragment is the internal positive control fragment of rDNA 28S D2/D3 resulting from the use of the universal primers. Other Meloidogyne spp. included in this study generated only one fragment of approximate to 500 bp in size. Using this approach, M. incognita, M. enterolobii, and M. javanica were identified and detected using DNA extracted directly from individual galls containing the Meloidogyne spp. at various stages of their life cycle. Moreover, the percentage of positive PCR amplification increased with nematode development and detection was usually easy after the late stage of the second-stage juvenile. The protocol was applied to galls from naturally infested roots and the results were found to be fast, sensitive, robust, and accurate. This present study is the first to provide a definitive diagnostic tool for M. incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls using a one-step multiplex PCR technique.