Lindergard Gabriella, Nydam Daryl V, Wade Susan E, Schaaf Stephanie L, Mohammed Hussni O
Section of Epidemiology, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Mol Diagn. 2003;7(3-4):147-53. doi: 10.1007/BF03260031.
The implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection of Cryptosporidium spp. oocysts in fecal samples from cattle.
Fecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the kappa-coefficient.
Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative for Cryptosporidium parvum by the flotation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol.
Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.
人畜共患原生动物的经济有效干预策略的实施依赖于灵敏且准确的诊断方法的开发。我们开展了一项研究以评估用于检测牛粪便样本中隐孢子虫属卵囊的聚合酶链反应（PCR）方法的准确性。
在粪便样本中加入不同数量的卵囊并确定该方法的检测限。评估了两种DNA提取方法：玻璃珠法和使用液氮的冻融法。开发了一种针对隐孢子虫小亚基核糖体RNA（SSU rRNA）和TRAP-C2基因的巢式PCR方法。使用kappa系数对在SSU rRNA和TRAP-C2位点对隐孢子虫属的诊断一致性进行了量化。
与冻融法相比，发现玻璃珠法是从隐孢子虫卵囊中提取DNA的更好方法（灵敏度分别为83%和100%）。玻璃珠法和冻融法的检测限较低，分别为每克粪便样本1个和10个卵囊。之前通过浮选浓缩和酶联免疫吸附测定法分类为小隐孢子虫阴性的46%的现场样本通过PCR方案显示有DNA。
用于SSU rRNA的引物比用于TRAP-C2基因的引物在产生扩增方面更成功，这使得前一种PCR方案成为检测现场样本中小隐孢子虫卵囊的首选方法。
