Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool.

Mio Ayana,Piet Cools,Roger Prichard,Johnny Vlaminck,Daniel Dana,Nour Rashwan,Abdissa Biruksew,Jaco J Verweij,Zeleke Mekonnen,Bruno Levecke,Cinzia Cantacessi

doi:10.1371/journal.pntd.0007778

Abstract

BackgroundA 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 findingsIn 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.ConclusionsThe 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.

Highlights

Soil-transmitted helminth (STH) infections are among the most common parasitic infections globally and affect more than a quarter of the world’s population, mainly poor populations intropical regions
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)
The choice of DNA extraction kit significantly affects the outcome of NAATs

Summary

Introduction

Soil-transmitted helminth (STH) infections are among the most common parasitic infections globally and affect more than a quarter of the world’s population, mainly poor populations in (sub)tropical regions. These infections are caused by intestinal helminths (worms) which excrete eggs through human stool, that contaminate soil in areas where sanitation is poor and in turn infect the human host orally or through skin contact. 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). We assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool

Methods

Results

Conclusion