Dual probe DNA capture for sensitive real-time PCR detection of Cryptosporidium and Giardia.
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.
University of Virginia, Division of Infectious Diseases and International Health, Charlottesville, VA 22908-1363, USA.
Suzanne
S
Stroup
Siripong
S
Tongjai
Ndealilia
N
Swai
Athanasia
A
Maro
Gibson
G
Kibiki
Eric R
ER
Houpt
D003457:Cryptosporidiosis / Q000175:diagnosis* / Q000469:parasitology; D003458:Cryptosporidium / Q000235:genetics*; D003967:Diarrhea / Q000469:parasitology; D005243:Feces / Q000469:parasitology; D005872:Giardia / Q000235:genetics*; D005873:Giardiasis / Q000175:diagnosis* / Q000469:parasitology; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques; D016053:RNA, Protozoan / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction
D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S