3 years ago

Evaluation of five automated and one manual methods for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid

Evaluation of five automated and one manual methods for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid
Molecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays. Methods In this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS® and Freedom EVO VacS, Tecan®), as compared to three other automated procedures (MagNAPure Compact, Roche®, BioRobot EZ1, Qiagen® and modified NucliSens easyMAG, bioMérieux®) and to the manual DNA extraction QIAamp DNA Mini kit (Qiagen®). Two Toxoplasma-PCR assays targeting the '529-bp' repeat DNA element were used, based upon dual hybridization (FRET) or hydrolysis (TaqMan) probes. A total of 1,296 PCRs were performed including 972 Toxoplasma-PCRs. Results We showed variable efficacy (4.2 to 79.2% positive results) among the DNA extraction procedures in isolating ≤5 T. gondii cells/mL in AF samples. Moreover, for a given DNA extraction method, variable results were obtained among the two Toxoplasma-PCR assays for detecting ≤5 T. gondii cells/mL: when using TaqMan PCR, all the automated systems yielded more than 60% of positive results. Nevertheless, when testing the DNA extracts in triplicate, four out of six extraction methods allowed a satisfactory detection of low amounts of T. gondii DNA (≥33% of positive results) independently of the PCR assay used. Conclusions Despite the influence of the subsequent PCR method used, this study should help microbiologists in the choice of DNA extraction methods for the detection of T. gondii in amniotic fluid. The extraction method should be checked as adequate to the PCR assay used.

Publisher URL: www.sciencedirect.com/science

DOI: S1198743X18300685

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.