Evaluation of Extraction Methods for Clinical Metagenomic Assay
(1) Background: Clinical metagenomics is a promising approach that helps to identify etiological agents in cases of unknown infections. For the efficient detection of an unknown pathogen, the extraction method must be carefully selected for the maximum recovery of nucleic acid from different microor...
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doaj-dce0dc966bd2434aa55acf32ef8566762020-11-25T03:09:19ZengMDPI AGMicroorganisms2076-26072020-07-0181128112810.3390/microorganisms8081128Evaluation of Extraction Methods for Clinical Metagenomic AssaySuha A. Farraj0Shreif A. El-Kafrawy1Taha A. Kumosani2Jehad M. Yousef3Esam I. Azhar4Biochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi ArabiaSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi ArabiaBiochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi ArabiaBiochemistry Department, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi ArabiaSpecial Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia(1) Background: Clinical metagenomics is a promising approach that helps to identify etiological agents in cases of unknown infections. For the efficient detection of an unknown pathogen, the extraction method must be carefully selected for the maximum recovery of nucleic acid from different microorganisms. The aim of this study was to evaluate different extraction methods that have the ability to isolate nucleic acids from different types of pathogens with good quality and quantity for efficient use in clinical metagenomic identification. (2) Methods: A mock sample spiked with five different pathogens was used for the comparative evaluation of different commercial extraction kits. Extracted samples were subjected to library preparation and run on MiSeq. The selected extraction method based on the outcome of the comparative evaluation was used subsequently for the nucleic acid isolation of all infectious agents in clinical respiratory samples with multiple infections. (3) Results: The protocol using the PowerViral<sup>®</sup> Environmental RNA-DNA Isolation Kit with a 5-min bead beating step achieved the best results with a low starting volume. The analysis of the tested clinical specimens showed the ability to successfully identify different types of pathogens. (4) Conclusions: The optimized extraction protocol in this study is recommended for clinical metagenomics application in specimens with multiple infections from different taxa.https://www.mdpi.com/2076-2607/8/8/1128clinical metagenomicsnucleic acid extractionmicroorganismsinfectious diseases |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Suha A. Farraj Shreif A. El-Kafrawy Taha A. Kumosani Jehad M. Yousef Esam I. Azhar |
spellingShingle |
Suha A. Farraj Shreif A. El-Kafrawy Taha A. Kumosani Jehad M. Yousef Esam I. Azhar Evaluation of Extraction Methods for Clinical Metagenomic Assay Microorganisms clinical metagenomics nucleic acid extraction microorganisms infectious diseases |
author_facet |
Suha A. Farraj Shreif A. El-Kafrawy Taha A. Kumosani Jehad M. Yousef Esam I. Azhar |
author_sort |
Suha A. Farraj |
title |
Evaluation of Extraction Methods for Clinical Metagenomic Assay |
title_short |
Evaluation of Extraction Methods for Clinical Metagenomic Assay |
title_full |
Evaluation of Extraction Methods for Clinical Metagenomic Assay |
title_fullStr |
Evaluation of Extraction Methods for Clinical Metagenomic Assay |
title_full_unstemmed |
Evaluation of Extraction Methods for Clinical Metagenomic Assay |
title_sort |
evaluation of extraction methods for clinical metagenomic assay |
publisher |
MDPI AG |
series |
Microorganisms |
issn |
2076-2607 |
publishDate |
2020-07-01 |
description |
(1) Background: Clinical metagenomics is a promising approach that helps to identify etiological agents in cases of unknown infections. For the efficient detection of an unknown pathogen, the extraction method must be carefully selected for the maximum recovery of nucleic acid from different microorganisms. The aim of this study was to evaluate different extraction methods that have the ability to isolate nucleic acids from different types of pathogens with good quality and quantity for efficient use in clinical metagenomic identification. (2) Methods: A mock sample spiked with five different pathogens was used for the comparative evaluation of different commercial extraction kits. Extracted samples were subjected to library preparation and run on MiSeq. The selected extraction method based on the outcome of the comparative evaluation was used subsequently for the nucleic acid isolation of all infectious agents in clinical respiratory samples with multiple infections. (3) Results: The protocol using the PowerViral<sup>®</sup> Environmental RNA-DNA Isolation Kit with a 5-min bead beating step achieved the best results with a low starting volume. The analysis of the tested clinical specimens showed the ability to successfully identify different types of pathogens. (4) Conclusions: The optimized extraction protocol in this study is recommended for clinical metagenomics application in specimens with multiple infections from different taxa. |
topic |
clinical metagenomics nucleic acid extraction microorganisms infectious diseases |
url |
https://www.mdpi.com/2076-2607/8/8/1128 |
work_keys_str_mv |
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