Department of Civil & Environmental Engineering, Arizona State University, Tempe, Arizona, USA.
Int J Infect Dis. 2011 Mar;15(3):e197-200. doi: 10.1016/j.ijid.2010.11.005. Epub 2010 Dec 23.
Globally, disinfectants are widely used to intervene in the dissemination of Cryptosporidium oocysts. However, extensive investigations of oocyst inactivation by various disinfectants are not feasible due to the limitations imposed by animal infectivity methods. Molecular techniques provide an alternative strategy; however, non-metabolic genes have been used as markers for determining viability/infectivity.
In this study we used amyloglucosidase (AG) - a metabolic protein - as a marker to determine viability/infectivity of Cryptosporidium. Oocysts were exposed to 6% hydrogen peroxide for 2min. Samples were analyzed by cell culture polymerase chain reaction (CC-PCR) using PCR primers specific for heat shock protein 70 (hsp70) and AG. Both target genes were amplified with the same level of intensity.
Based on the results it can be concluded that AG is a valid target for the study of environmental survival and for the evaluation of the efficacy of microbicides against Cryptosporidium using molecular and cellular assays. Comparison of the CC-PCR assay and mouse infectivity assay showed a fairly good correlation under these test conditions.
Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.
在全球范围内，消毒剂被广泛用于干预隐孢子虫卵囊的传播。然而，由于动物感染性方法的限制，对各种消毒剂对卵囊灭活的广泛调查是不可行的。分子技术提供了一种替代策略；然而，非代谢基因已被用作确定生存能力/感染性的标记物。
在这项研究中，我们使用了淀粉酶（AG） - 一种代谢蛋白 - 作为标记物来确定隐孢子虫的生存能力/感染性。卵囊用 6%过氧化氢暴露 2 分钟。使用针对热休克蛋白 70（hsp70）和 AG 的 PCR 引物通过细胞培养聚合酶链反应（CC-PCR）分析样品。两个靶基因以相同的强度水平扩增。
根据结果可以得出结论，AG 是研究环境生存能力以及使用分子和细胞测定评估抗隐孢子虫杀菌剂功效的有效标记物。CC-PCR 测定法和小鼠感染性测定法的比较在这些测试条件下显示出相当好的相关性。
结果表明，CC-PCR 测定法是小鼠感染性测定法的有效且具有成本效益的替代方法。
