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

Autor:	Piet Cools, Roger K. Prichard, Zeleke Mekonnen, Bruno Levecke, Abdissa Biruksew, Nour Rashwan, Mio Ayana, Johnny Vlaminck, Daniel Dana, Jaco J. Verweij
Jazyk:	angličtina
Rok vydání:	2019
Předmět:	Ancylostomatoidea 0301 basic medicine Ascaris Lumbricoides Nematoda Physiology Necator americanus RC955-962 Helminthiasis NECATOR-AMERICANUS law.invention Feces Soil chemistry.chemical_compound 0302 clinical medicine law Arctic medicine. Tropical medicine Medicine and Health Sciences Child DNA extraction Polymerase chain reaction Ascariasis biology Organic Compounds Ascaris Eukaryota Necator NEMATODE Body Fluids TRICHURIS-TRICHIURA INFECTIONS Chemistry Blood Trichuris PCR DOGS Infectious Diseases Real-time polymerase chain reaction Molecular Diagnostic Techniques Helminth Infections Ancylostoma duodenale Child Preschool Physical Sciences Anatomy Public aspects of medicine RA1-1270 Research Article Neglected Tropical Diseases ANCYLOSTOMA-CEYLANICUM Ancylostoma Adolescent Preservation Biological 030231 tropical medicine DUODENALE DIAGNOSIS Sensitivity and Specificity DNA sequencing Necatoriasis 03 medical and health sciences Extraction techniques Helminths parasitic diseases Parasitic Diseases Animals Humans Trichuriasis Parasite Egg Count Chromatography Ethanol EGGS Organic Chemistry Organisms Chemical Compounds Public Health Environmental and Occupational Health Biology and Life Sciences DNA Tropical Diseases biology.organism_classification Invertebrates Research and analysis methods FECAL SAMPLES 030104 developmental biology Soil-Transmitted Helminthiases chemistry Hookworms Alcohols Trichuris trichiura Preservatives
Zdroj:	PLoS Neglected Tropical Diseases, Vol 13, Iss 10, p e0007778 (2019) PLOS NEGLECTED TROPICAL DISEASES BASE-Bielefeld Academic Search Engine PLoS Neglected Tropical Diseases
ISSN:	1935-2735 1935-2727
Popis:	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 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. 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. Author summary 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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3eb3a5e225c624d012311b8204f5d220 https://doaj.org/article/c69a4a4354714974a628557b87ddd08f Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
Nepřihlášeným uživatelům se plný text nezobrazuje	K zobrazení výsledku je třeba se přihlásit.