一种用于鉴定五种主要根结线虫种类的聚合酶链反应方法。
A polymerase chain reaction method for identification of five major meloidogyne species.
作者信息
Powers T O, Harris T S
出版信息
J Nematol. 1993 Mar;25(1):1-6.
A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.
开发了一种用于区分南方根结线虫、花生根结线虫、爪哇根结线虫、北方根结线虫和奇氏根结线虫的聚合酶链反应（PCR）方法。将单个幼虫在一滴水 中弄破，然后直接加入到微量离心管中的PCR反应混合物中。引物退火位点位于编码细胞色素氧化酶亚基II的线粒体基因的3'部分和16S rRNA基因中。PCR扩增后，检测到三种大小的片段。南方根结线虫和爪哇根结线虫的反应产生一个1.7 kb的片段；花生根结线虫的反应产生一个1.1 kb的片段；北方根结线虫和奇氏根结线虫的反应产生一个0.52 kb的片段。用限制性内切酶消化扩增产物可区分扩增产物大小相同的物种。用Dra I消化0.52 kb的扩增产物，在奇氏根结线虫中产生特征性的三条带模式，而在北方根结线虫中产生两条带模式。用Hinf I消化1.7 kb的片段，在爪哇根结线虫中产生两条带模式，而在南方根结线虫中产生三条带模式。对来自马里兰根结线虫、纳氏根结线虫和纳塔尔根结线虫单个分离物的幼虫DNA进行扩增和消化表明，该引物组的诊断应用可能扩展到较少见的根结线虫物种。
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