Dual probe DNA capture for sensitive real-time PCR detection of Cryptosporidium and Giardia

Mol Cell Probes. 2012 Apr;26(2):104-6. doi: 10.1016/j.mcp.2011.12.003. Epub 2011 Dec 30.

Abstract

Nucleic acid amplification for the enteropathogens Cryptosporidium and Giardia is complicated by low target template concentrations and PCR inhibitors. In this work we designed dual capture oligonucleotides for both Cryptosporidium and Giardia 18S rRNA targets which when utilized during DNA extraction from stool improved the limit of detection of our multiplex PCR assay by 1-2 logs, to as little as 10 cysts. When applied to clinical specimens, the method improved the real-time PCR C(T) by an average of 10.7 ± 9.7 cycles. This work provides a highly sensitive protocol for Cryptosporidium and Giardia when limit of detection is of utmost importance.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cryptosporidiosis / diagnosis*
  • Cryptosporidiosis / parasitology
  • Cryptosporidium / genetics*
  • Diarrhea / parasitology
  • Feces / parasitology
  • Giardia / genetics*
  • Giardiasis / diagnosis*
  • Giardiasis / parasitology
  • Limit of Detection
  • Molecular Diagnostic Techniques
  • RNA, Protozoan / genetics*
  • RNA, Ribosomal, 18S / genetics*
  • Real-Time Polymerase Chain Reaction*

Substances

  • RNA, Protozoan
  • RNA, Ribosomal, 18S