Open AccessComparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool
Author(s) -
Mio Ayana,
Piet Cools,
Zeleke Mekonnen,
Abdissa Biruksew,
Daniel Dana,
Nour Rashwan,
Roger K. Prichard,
Johnny Vlaminck,
Jaco J. Verweij,
Bruno Levecke
Publication year - 2019
Publication title -
plos neglected tropical diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.99
H-Index - 135
eISSN - 1935-2735
pISSN - 1935-2727
DOI - 10.1371/journal.pntd.0007778
Subject(s) - necator americanus , ascaris lumbricoides , dna extraction , ancylostoma duodenale , biology , trichuris trichiura , helminths , dna , polymerase chain reaction , microbiology and biotechnology , genetics , immunology , gene
Background 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. Methodology and principal findings 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 RNA later 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. Conclusions 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.