Ey P L, Andrews R H, Mayrhofer G
Department of Microbiology and Immunology, University of Adelaide, South Australia.
Parasitology. 1993 May;106 ( Pt 4):347-56. doi: 10.1017/s0031182000067081.
The polymerase chain reaction (PCR) has been used to amplify in vitro a semi-conserved region of a gene encoding an M(r) 68-72,000 surface antigen of Giardia intestinalis trophozoites. Using primers specific for conserved nucleotide sequences identified within the promoter-distal portion of two homologous genes (tsp11 and tsa417) cloned previously from the G. intestinalis isolates Ad-1 (from Australia) and WB (from Afghanistan), a single PCR-amplified DNA fragment of the expected size (0.52 kilobases) was obtained in high yield from either purified DNA or whole trophozoites of the Ad-1 isolate and from every 1 to 9 other axenic G. intestinalis isolates belonging to genetic groups I and II (defined previously on the basis of allozyme electrophoresis data--Andrews et al. 1989). Discernible product was recovered from as few as 2-4 trophozoites. In contrast, 6 G. intestinalis isolates that were assigned by allozymic analysis to genetic groups III/IV yielded small amounts of a 0.37-kilobase (kb) amplification product (with evidence in some samples of an additional 0.4 or 0.18 kb fragment) but no 0.52 kb product. Two animal-derived isolates of G. duodenalis (one from an Australian native rodent, Notomys alexis, the other from a domestic cat) also yielded a single 0.37 kb PCR-amplified fragment, whereas an isolate from another cat produced a 0.34 kb fragment. No product was recovered from G. muris, a morphologically distinct species of Giardia. The results demonstrate that different genotypes of G. duodenalis can be distinguished using this assay and that it is diagnostic for isolates belonging to two major clusters (groups I/II and III/IV) of G. intestinalis. The amplified DNA segment appears to be relatively conserved among group I and group II isolates of G. intestinalis. A related but clearly distinct sequence seems to be conserved among group III/IV isolates of G. intestinalis and some isolates of G. duodenalis.
聚合酶链反应（PCR）已用于体外扩增编码贾第虫滋养体68 - 72,000 M（r）表面抗原的基因的半保守区域。使用针对先前从贾第虫分离株Ad - 1（来自澳大利亚）和WB（来自阿富汗）克隆的两个同源基因（tsp11和tsa417）的启动子远端部分中鉴定出的保守核苷酸序列的引物，从Ad - 1分离株的纯化DNA或整个滋养体以及属于遗传组I和II（先前根据等位酶电泳数据定义 - Andrews等人，1989）的每1至9个其他无菌贾第虫分离株中高产获得了预期大小（0.52千碱基）的单个PCR扩增DNA片段。从少至2 - 4个滋养体中可回收可辨别的产物。相比之下，通过等位酶分析分配到遗传组III / IV的6个贾第虫分离株产生少量的0.37千碱基（kb）扩增产物（在一些样品中有另外0.4或0.18 kb片段的证据），但没有0.52 kb产物。两个源自动物的十二指肠贾第虫分离株（一个来自澳大利亚本土啮齿动物Notomys alexis，另一个来自家猫）也产生了单个0.37 kb的PCR扩增片段，而来自另一只猫的分离株产生了0.34 kb片段。从形态上不同的贾第虫物种鼠贾第虫中未回收产物。结果表明，使用该测定法可以区分十二指肠贾第虫的不同基因型，并且它对属于贾第虫两个主要簇（I / II组和III / IV组）的分离株具有诊断性。扩增的DNA片段在贾第虫的I组和II组分离株中似乎相对保守。一个相关但明显不同的序列似乎在贾第虫的III / IV组分离株和一些十二指肠贾第虫分离株中保守。
