umu.sePublications
Change search
ReferencesLink to record
Permanent link

Direct link
Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Show others and affiliations
2015 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 64, 1053-1062 p.Article in journal (Refereed) Published
Abstract [en]

Although PCR offers the potential for sensitive detection of parasites:there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

Place, publisher, year, edition, pages
Microbiology Society , 2015. Vol. 64, 1053-1062 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-111507DOI: 10.1099/jmm.0.000129ISI: 000363356800016PubMedID: 26296348OAI: oai:DiVA.org:umu-111507DiVA: diva2:875357
Available from: 2015-12-01 Created: 2015-11-13 Last updated: 2015-12-01Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Forsell, JoakimKoskiniemi, SatuHedberg, IdaEdebro, HelenEvengård, BirgittaGranlund, Margareta
By organisation
Clinical BacteriologyInfectious Diseases
In the same journal
Journal of Medical Microbiology
Microbiology in the medical area

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 30 hits
ReferencesLink to record
Permanent link

Direct link