Shields Joan M, Olson Betty H
Department of Environmental Analysis and Design, School of Social Ecology, University of California, Irvine, California 92697-7070, USA.
Appl Environ Microbiol. 2003 Aug;69(8):4662-9. doi: 10.1128/AEM.69.8.4662-4669.2003.
We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.
我们开发了一种替代性的巢式聚合酶链反应-限制性片段长度多态性（RFLP）方法，用于检测环境样本中的卡耶塔环孢子虫，该方法无需显微镜检查。使用限制性内切酶AluI的RFLP方法，可将卡耶塔环孢子虫的扩增靶序列与可能发生交叉反应的序列区分开来。该新方法用于重新检测一部分（180份中的121份）地表水样本。以前使用CYCF3E和CYCR4B引物（33）以及MnlI进行RFLP时呈阳性的样本，使用新引物进行PCR时也呈阳性；然而，它们的RFLP结果为阴性。通过对这些扩增子进行测序，我们证实，虽然其中两份很可能是其他环孢子虫物种，但不是卡耶塔环孢子虫。我们能够检测到接种到从10升地表水中絮凝得到的高压灭菌沉淀物中的低至一个卵囊。该新方法对环境微生物学家和公共卫生实验室应该非常有用。
