title: A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp.
abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3'-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5'-end forms a hairpin structure. A fluorescent dye is attached to 5'-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3' dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000-0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments.
authors.affiliation: Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Waterborne Disease Prevention Branch, 1600 Clifton Road, Atlanta, GA 30329, USA. jin2@cdc.gov
authors.forename: N
authors.initials: N
authors.lastname: Jothikumar
authors.forename: Vincent R
authors.initials: VR
authors.lastname: Hill
mesh_terms: D001483:Base Sequence; D016785:Cryptosporidium parvum / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D004579:Electron Transport / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000737:chemistry; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D033661:Oocysts / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D005456:Fluorescent Dyes