Gasser R B, Zhu X, Caccio S, Chalmers R, Widmer G, Morgan U M, Thompson R C, Pozio E, Browning G F
Department of Veterinary Science, The University of Melbourne, Victoria, Australia.
Electrophoresis. 2001 Feb;22(3):433-7. doi: 10.1002/1522-2683(200102)22:3<433::AID-ELPS433>3.0.CO;2-K.
Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.
利用核糖体RNA小亚基（SSU）和热休克蛋白70基因（HSP70）的核DNA区域，建立了聚合酶链反应（PCR）偶联单链构象多态性（SSCP）方法，用于微小隐孢子虫的基因分型。通过PCR从反刍动物或人类宿主的卵囊中提取的DNA中扩增这些区域（单独或在多重反应中），然后变性并在突变检测增强（非变性）凝胶基质中进行电泳。SSCP产生的单链图谱能够明确鉴定/区分微小隐孢子虫的两种常见基因型（人类基因型1和牛基因型2），并直接显示一些样本中的序列变异性，反映种群变异。由于基于SSU和HSP70序列数据，这些基因型被认为是关系最为密切的，这些发现以及针对猫隐孢子虫（来自猫）、蛇隐孢子虫（来自蛇）和贝氏隐孢子虫（来自鸟类）的其他初步结果表明，所建立的SSCP方法可用于鉴定隐孢子虫目前已确认的任何基因型和种类。
