Homan W L, Gilsing M, Bentala H, Limper L, van Knapen F
Microbiological Laboratory of Health Protection, National Institute of Public Health and the Environment, Bilthoven, The Netherlands.
Parasitol Res. 1998 Sep;84(9):707-14. doi: 10.1007/s004360050474.
The development of a polymerase chain reaction (PCR) based fingerprinting method for the characterization of Giardia duodenalis isolates is described. The method uses three different PCRs; one is specific for the A ("Polish") major group of G. duodenalis isolates, another is specific for the B ("Belgian") group of isolates; and one amplifies a fragment of the glutamate dehydrogenase gene present in all G. duodenalis isolates. The PCRs perform highly sensitively on DNA from cultured trophozoites. Isolates were further characterized by restriction-fragment-length polymorphism (RFLP) analysis of the PCR products. In this way, representative isolates from the A and B groups could be grouped together into a number of subgroups. The stability of the genotypes with time and the reproducibility of the two methods were tested on cloned and subcloned lines from a number of isolates and proved to be highly satisfactory. The PCR/RFLP method was evaluated on cysts derived from a number of human patients. It is concluded that the PCR fingerprinting method described in this paper provides a reliable characterization method for Giardia isolates and has the potential to be used as a direct method of typing G. duodenalis cysts from feces.
本文描述了一种基于聚合酶链反应（PCR）的指纹图谱方法的开发，用于鉴定十二指肠贾第鞭毛虫分离株的特征。该方法使用三种不同的PCR；一种对十二指肠贾第鞭毛虫分离株的A（“波兰”）主要群体具有特异性，另一种对分离株的B（“比利时”）群体具有特异性；还有一种扩增所有十二指肠贾第鞭毛虫分离株中存在的谷氨酸脱氢酶基因片段。这些PCR对培养的滋养体DNA具有高度敏感性。通过对PCR产物进行限制性片段长度多态性（RFLP）分析，进一步鉴定分离株。通过这种方式，A组和B组的代表性分离株可以被归为多个亚组。在来自多个分离株的克隆和亚克隆系上测试了基因型随时间的稳定性以及这两种方法的可重复性，结果证明非常令人满意。对来自多名人类患者的囊肿进行了PCR/RFLP方法评估。得出的结论是，本文所述的PCR指纹图谱方法为贾第鞭毛虫分离株提供了一种可靠的鉴定方法，并且有可能用作直接鉴定粪便中十二指肠贾第鞭毛虫囊肿类型的方法。
