MacMillan Keith, Blok Vivian, Young Iain, Crawford John, Wilson Michael J
School of Biological Sciences, University of Aberdeen, Aberdeen AB24 3UU, Scotland, UK.
Int J Parasitol. 2006 Dec;36(14):1453-61. doi: 10.1016/j.ijpara.2006.08.005. Epub 2006 Sep 15.
Phasmarhabditis hermaphrodita is a nematode parasite that infects and kills several species of slugs. The nematode is produced commercially as a biological control agent for slug pests of agriculture and horticulture. Given the difficulties of distinguishing this species from other nematode species in soil samples, very little is known about its natural ecology or its behaviour and persistence following application for biological control. Here we describe a method to quantify P. hermaphrodita in soil samples based on real time PCR. We designed primers and a dual labelled fluorescent probe that can be used to quantify numbers of P. hermaphrodita and which is capable of distinguishing this species from the morphologically identical Phasmarhabditis neopapillosa. We compared different methods whereby the entire nematode community is extracted prior to DNA extraction, and three methods to extract DNA directly from soil samples. Both nematode extraction and DNA extraction from large (10 g) samples of soil gave reliable estimates of nematode numbers, but methods which extracted DNA from small (1g or less) soil samples substantially underestimated numbers. However, direct extraction of DNA from soils may overestimate numbers of live nematodes as DNA from dead nematodes was found to persist in soil for at least 6 days. The technique could be modified for detection and quantification of all soil borne parasitic nematodes.
雌雄同体细线虫是一种能感染并杀死多种蛞蝓的线虫寄生虫。这种线虫作为农业和园艺蛞蝓害虫的生物防治剂进行商业化生产。鉴于在土壤样本中难以将该物种与其他线虫物种区分开来，人们对其自然生态、施用生物防治后的行为及持久性知之甚少。在此，我们描述一种基于实时聚合酶链反应（PCR）对土壤样本中的雌雄同体细线虫进行定量的方法。我们设计了引物和一种双标记荧光探针，可用于定量雌雄同体细线虫的数量，并能将该物种与形态上相同的新乳头细线虫区分开来。我们比较了在DNA提取前提取整个线虫群落的不同方法，以及直接从土壤样本中提取DNA的三种方法。从大的（10克）土壤样本中提取线虫和DNA都能可靠地估计线虫数量，但从小的（1克或更少）土壤样本中提取DNA的方法会大幅低估数量。然而，直接从土壤中提取DNA可能会高估活线虫的数量，因为发现死线虫的DNA在土壤中至少能持续存在6天。该技术可进行修改，用于检测和定量所有土壤传播的寄生线虫。
