Waldron L S, Ferrari B C, Gillings M R, Power M L
Biological Sciences, Macquarie University, Sydney NSW 2109, Australia.
Appl Environ Microbiol. 2009 Jan;75(1):108-12. doi: 10.1128/AEM.01341-08. Epub 2008 Oct 31.
Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.
人类隐孢子虫病的有效管理需要高效的方法来检测和鉴定隐孢子虫分离株的种类。对于隐孢子虫病的诊断评估，将分离株鉴定到种水平并非常规操作，这导致了引起人类疾病的隐孢子虫种类的流行病学存在不确定性。我们开发了一种快速可靠的方法，利用18S rRNA基因的末端限制性片段长度多态性（T-RFLP）分析，从人类粪便样本中鉴定隐孢子虫卵囊的种类。该方法产生了目标物种特有的诊断片段。对一组先前鉴定的物种分离株进行了盲法测试以验证该方法，该方法在每种情况下都确定了正确的物种身份。对于添加了已知数量的人隐孢子虫和微小隐孢子虫卵囊的样本所获得的T-RFLP图谱产生了两个预期的诊断峰。单个物种的检测限为总DNA的1%。这是T-RFLP在原生动物中的首次应用，我们开发的方法是一种快速、可重复且经济高效的物种鉴定方法。
