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- Title
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool.
- Authors
Ayana, Mio; Cools, Piet; Mekonnen, Zeleke; Biruksew, Abdissa; Dana, Daniel; Rashwan, Nour; Prichard, Roger; Vlaminck, Johnny; Verweij, Jaco J.; Levecke, Bruno
- Abstract
A DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool. In a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNAlater and were stored at 4°C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives. The choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs. DNA-based tools are increasingly being used for the diagnosis of intestinal worm infections in both clinical and research laboratories. However, recovering DNA from intestinal worm eggs in stool remains a challenge since this DNA is protected by a very rigid egg shell. Furthermore, stool contains inhibitors that can affect test results and these should be removed during DNA extraction. Prior to DNA extraction, samples are often preserved, but the impact of the type of preservatives and the duration of preservation remains poorly studied. In the present study, we assessed the impact of four DNA extraction and three preservation protocols on the downstream performance of a DNA-based diagnostic tool for intestinal worms. We found significant differences in DNA recovery across the DNA and preservation protocols, but DNA from worm eggs in stool proved to be stable over time in all preservatives.
- Subjects
NUCLEIC acid isolation methods; NUCLEIC acid amplification techniques; HOOKWORM disease; DNA; WORM eggs; ASCARIS lumbricoides; GENE amplification
- Publication
PLoS Neglected Tropical Diseases, 2019, Vol 13, Issue 10, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0007778