title: Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR.
abstract: The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.
authors.affiliation: Department of Genetics, University of Szczecin, al. Piastów 40b, 71-065 Szczecin, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Adamska
authors.forename: A
authors.initials: A
authors.lastname: Leońska-Duniec
authors.forename: A
authors.initials: A
authors.lastname: Maciejewska
authors.forename: M
authors.initials: M
authors.lastname: Sawczuk
authors.forename: B
authors.initials: B
authors.lastname: Skotarczak
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology; D017931:DNA Primers; D016054:DNA, Protozoan / Q000302:isolation & purification*; D005615:Freezing; D016829:Giardia lamblia / Q000235:genetics* / Q000302:isolation & purification; D005873:Giardiasis / Q000662:veterinary; D009584:Nitrogen; D016133:Polymerase Chain Reaction / Q000379:methods; D015800:Protozoan Proteins / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D009584:Nitrogen