Pujol-Riqué Marc, Derouin Francis, Garcia-Quintanilla Alberto, Valls M E, Miró J M, Jiménez De Anta M T
Servei de Microbiologia.
Laboratoire de Parasitologie-Mycologie, Hospital Saint-Louis, Paris, France.
J Med Microbiol. 1999 Sep;48(9):857-862. doi: 10.1099/00222615-48-9-857.
The aims of the present study were to design an easy and sensitive DNA amplification method for detection of Toxoplasma gondii with low risk of accidental contamination, and to find a rapid method for purification of clinical samples containing potential inhibitors of the amplification reaction. With a pair of primers amplifying a 619-bp fragment of the B1 gene of this parasite it was possible to detect DNA equivalent to 10 parasites. When a third primer was added to the same tube, sensitivity increased to 0.1 parasite. In a comparison of different DNA purification methods, the High Pure PCR Template Preparation Kit (Boehringer Mannheim, Germany) gave the best results. With this purification method and the one-tube hemi-nested PCR, T. gondii DNA was detected in 14 (87.5%) of 16 clinical specimens (amniotic fluid, broncho-alveolar lavage, bone marrow, blood, liver biopsy) in which the parasite was demonstrated by cell culture.
本研究的目的是设计一种简便且灵敏的DNA扩增方法，用于检测弓形虫，同时降低意外污染风险，并找到一种快速方法来纯化含有潜在扩增反应抑制剂的临床样本。使用一对引物扩增该寄生虫B1基因的619bp片段，能够检测到相当于10个寄生虫的DNA。当向同一管中加入第三种引物时，灵敏度提高到0.1个寄生虫。在不同DNA纯化方法的比较中，高纯PCR模板制备试剂盒（德国宝灵曼公司）效果最佳。采用这种纯化方法和单管半巢式PCR，在16份临床标本（羊水、支气管肺泡灌洗、骨髓、血液、肝活检）中有14份（87.5%）检测到弓形虫DNA，这些标本经细胞培养证实存在该寄生虫。
