Bretagne S, Guillou J P, Morand M, Houin R
Laboratoire de Parasitologie, Faculté de Médecine, Créteil, France.
Parasitology. 1993 Feb;106 ( Pt 2):193-9. doi: 10.1017/s0031182000074990.
In order to identify Echinococcus multilocularis DNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol-chloroform extraction and a purification step on a matrix (Prep-A-Gene). The target sequence for amplification was the E. multilocularis U1 snRNA gene. PCR products were indistinguishable for 32 different E. multilocularis isolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, including E. granulosus. The sensitivity of amplification was monitored by the addition of E. multilocularis DNA or eggs to faeces free of E. multilocularis and was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which no E. multilocularis adult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouring E. multilocularis adult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosus and E. multilocularis), or to DNA variation between different isolates of E. multilocularis. Further work in the field should be initiated in order to confirm these results.
为了从狐狸粪便中鉴定出多房棘球绦虫DNA用于流行病学研究，我们开发了一种新方法来制备适合PCR扩增的DNA。从狐狸粪便中分离DNA是按照一种新程序进行的，该程序包括在氢氧化钾中裂解、酚-氯仿提取以及在基质（Prep-A-Gene）上进行纯化步骤。扩增的靶序列是多房棘球绦虫U1 snRNA基因。对于32种不同的多房棘球绦虫分离株，PCR产物无法区分，在用来自其他绦虫物种（包括细粒棘球绦虫）的DNA进行溴化乙锭染色后未观察到信号。通过向不含多房棘球绦虫的粪便中添加多房棘球绦虫DNA或虫卵来监测扩增的灵敏度，估计灵敏度为每4克粪便1个虫卵。将PCR产物转移到尼龙膜上，并用内部寡核苷酸探针进行杂交以确认结果。对在法国东部弗朗什-孔泰射杀的狐狸的29份粪便样本进行了检测。在10份尸检后未观察到多房棘球绦虫成虫的狐狸样本中，有7份PCR呈阳性，表明PCR检测比显微镜检查更灵敏。在19份含有多房棘球绦虫成虫的狐狸样本中，有18份PCR呈阳性。其余PCR阴性样本可能是由于成虫物种（细粒棘球绦虫和多房棘球绦虫）鉴定错误，或者是由于多房棘球绦虫不同分离株之间的DNA变异。为了证实这些结果，应在该领域开展进一步的工作。
