Reproductive tract microbiome during pregnancy – DNA extraction methods, metagenomic analysis and identification of Candida albicans
Ladataan...
2.26 MB
suljettu
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
Lataukset2
Pysyvä osoite
Verkkojulkaisu
DOI
Tiivistelmä
Vaginal microbiota has low microorganism diversity and biomass compared to other body sites. This challenges the analysis path from DNA extraction to final stage of metagenomics. Selecting a suitable DNA extraction method is the first and important phase to preserve the comparability of the samples and to prevent possible bias in the microorganism composition. The aim of this study was to find an optimal DNA extraction method for the vaginal microbiota samples. In addition, the aim was to establish quantitative PCR (qPCR) method to detect Candida albicans from the microbiota samples.
Four different commercial DNA extraction kits were selected for the evaluation. DNA was extracted from 40 vaginal microbiota samples collected from pregnant women. The quality of the DNA extraction was evaluated by metagenomics analysis from a subset of five microbiota samples of which DNA was extracted with four kits. The presence of C. albicans was analysed with the qPCR protocol developed from 140 DNA extractions from 40 microbiota samples.
The DNA extraction method had a significant impact on the quality and quantity of the extracted DNA (p<0.0001, Friedmans test). Metagenomic analysis revealed that microbiota composition varied between individuals, but all samples from the same individual had similar profiles despite the DNA extraction kit used. C. albicans qPCR was successfully established and was sensitive for C. albicans detection. C. albicans was detected in 15% (6/40) of the microbiota samples. Although differences were detected between DNA extraction kit results, the effect on metagenomic results was small. In addition, the prevalence of C. albicans was high in the vaginal microbiota samples as expected.