The Sensitivity of PCR Detection of Cryptosporidium Oocysts in Fecal Samples Using Two DNA Extraction Methods

被引：0

作者：

Gabriella Lindergard

Daryl V. Nydam

Susan E. Wade

Stephanie L. Schaaf

Hussni O. Mohammed

机构：

[1] Dept. of Pop. Med. and Diagn. Sci., College of Veterinary Medicine, Cornell University, Ithaca

[2] Section of Parasitology, College of Veterinary Medicine, Cornell University, Ithaca, NY

来源：

Molecular Diagnosis | 2003年 / 7卷 / 3期

关键词：

Fecal Sample; Glass Bead; Cryptosporidium Parvum; Cryptosporidium Oocyst; Cryptosporidium Infection;

D O I：

10.1007/BF03260031

中图分类号：

学科分类号：

摘要：

Background: 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. Methods: 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 κ-coefficient. Results: 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 floatation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol. Conclusion: 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.

引用

页码：147 / 153

页数：6

共 29 条

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