[Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction].
This study was aimed to adapt a sensitive DNA extraction protocol in stool samples for real-time polymerase chain reaction (PCR) detection of Entamoeba histolytica which causes important morbidity and mortality worldwide. Stool extraction is a problematic step and has direct effects on PCR sensitivity. In order to improve the sensitivity of E.histolytica detection by real-time PCR, "QIAamp DNA stool minikit (Qiagen, Germany)" was modified by adding an overnight incubation step with proteinase K and sodium dodecyl sulfate (SDS) in this study. Three different extraction methods [(1) original method, (2) cetyltrimethyl-ammonium bromide (CTAB) method, (3) modified method] were evaluated for effects on sensitivity in real-time quantitative PCR (Artus RealArt TM E.histolytica RG PCR Kit, Qiagen Diagnostics, Germany). For this purpose, several concentrations of standard E.histolytica DNA were spiked in parasite-free stool samples and three different extraction protocols were performed. Detection sensitivities of "QIAamp DNA stool minikit" was found 5000 copies/ml and of CTAB method was found 500 copies/ml. Detection sensitivity of the extraction was improved to 5 copies/mL by modified "QIAamp DNA stool minikit" protocol. Since detection sensitivities of nucleic acid extraction protocols from stool samples directly affect the sensitivity of PCR amplification, different extraction protocols for different microorganisms should be evaluated.
Hacettepe Üniversitesi Tıp Fakültesi, Tıbbi Mikrobiyoloji Anabilim Dalı, Ankara, Türkiye. apinar@hacettepe.edu.tr
Ahmet
A
Pınar
Yakut
Y
Akyön
Alpaslan
A
Alp
Sibel
S
Ergüven
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D016054:DNA, Protozoan / Q000302:isolation & purification*; D004404:Dysentery, Amebic / Q000175:diagnosis* / Q000469:parasitology; D019286:Endopeptidase K / Q000737:chemistry; D004748:Entamoeba histolytica / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D002593:Cetrimonium Compounds; D016054:DNA, Protozoan; D012967:Sodium Dodecyl Sulfate; D019286:Endopeptidase K; D000077286:Cetrimonium