Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR
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. P...
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doaj-273ea8bdd155432e9ead6a58467b219f2021-03-02T01:05:10ZengEDP SciencesParasite1252-607X1776-10422010-12-0117429930510.1051/parasite/2010174299parasite2010174p299Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCRAdamska M.Leońska-Duniec A.Maciejewska A.Sawczuk M.Skotarczak B.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 ˚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 ˚C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of β-giardin gene fragment and CT 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 μl 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.http://dx.doi.org/10.1051/parasite/2010174299DNA extractionGiardia intestinalisPCRreal time PCR |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Adamska M. Leońska-Duniec A. Maciejewska A. Sawczuk M. Skotarczak B. |
spellingShingle |
Adamska M. Leońska-Duniec A. Maciejewska A. Sawczuk M. Skotarczak B. Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR Parasite DNA extraction Giardia intestinalis PCR real time PCR |
author_facet |
Adamska M. Leońska-Duniec A. Maciejewska A. Sawczuk M. Skotarczak B. |
author_sort |
Adamska M. |
title |
Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR |
title_short |
Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR |
title_full |
Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR |
title_fullStr |
Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR |
title_full_unstemmed |
Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR |
title_sort |
comparison of efficiency of various dna extraction methods from cysts of giardia intestinalis measured by pcr and taqman real time pcr |
publisher |
EDP Sciences |
series |
Parasite |
issn |
1252-607X 1776-1042 |
publishDate |
2010-12-01 |
description |
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 ˚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 ˚C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of β-giardin gene fragment and CT 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 μl 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. |
topic |
DNA extraction Giardia intestinalis PCR real time PCR |
url |
http://dx.doi.org/10.1051/parasite/2010174299 |
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