Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications

Kajiura, Lauren; Stewart, Scott; Dresios, John; Uyehara, Catherine F. T.

doi:10.7171/jbt.15-2604-002

Cited by 9 publications

(7 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The first protocol was conducted using the PowerViral ® Environmental RNA/DNA Isolation Kit (MoBio samples), and the other protocol was the "Copurification" protocol of the ZymoBIOMICS™ DNA/RNA Miniprep Kit (Zymo samples). Although we used the approach of Leite et al [30], who performed 2 cycles of 25-s agitation with a 5-s interval break, which they recommended to avoid possible degradation of nucleic acids and which was also reported by others [31,32], we improved the results of the MoBio and Zymo assays by performing the bead beating step at 4 • C and increasing the time to 5 min. Apart from the significant differences for the 5-min MoBio protocol compared with the 5-min Zymo protocol, the volume of the starting material for the MoBio extraction was 20% less than that for the Zymo kits (200 µL vs. 250 µL, respectively), and the number of steps of MoBio was fewer than that of Zymo, reducing the possibility of human error or contamination.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Extraction Methods for Clinical Metagenomic Assay

et al. 2020

View full text Add to dashboard Cite

(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® 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.

show abstract

Section: Discussionmentioning

confidence: 99%

Evaluation of Extraction Methods for Clinical Metagenomic Assay

et al. 2020

View full text Add to dashboard Cite

show abstract

“…For Staphylococcus , as with other organisms that possess thick cell walls, specialized DNA-extraction methods are required 39–42. If these methods are used, it has been shown that increased detection of Staphylococcus comes at the expense of microbial organisms with a fragile cell wall and viruses 43,44. Currently, there is no commercial nucleic acid-extraction kit that can simultaneously extract Gram-positive, Gram-negative, and viruses from the same sample.…”

Section: Discussionmentioning

confidence: 99%

“…Currently, there is no commercial nucleic acid-extraction kit that can simultaneously extract Gram-positive, Gram-negative, and viruses from the same sample. Kajiura et al44 proposed a method for achieving this goal through techniques that prevent the loss of smaller viral particles while still extracting Gram-positive bacteria. This has been successfully used on 300 archived clinical samples of respiratory origin.…”

Section: Discussionmentioning

confidence: 99%

<p>Phenotyping and outcomes of hospitalized COPD patients using rapid molecular diagnostics on sputum samples</p>

Alotaibi¹,

Chen²,

Hollander³

et al. 2019

COPD

View full text Add to dashboard Cite

BackgroundEtiologies of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are heterogeneous. We phenotyped severe AECOPD based on molecular pathogen detection of sputum samples collected at hospitalization of COPD patients and determined their outcomes.MethodsWe phenotyped 72 sputum samples of COPD patients who were hospitalized with a primary diagnosis of AECOPD using a molecular array that detected common bacterial and viral respiratory pathogens. Based on these results, the patients were classified into positive or negative pathogen groups. The pathogen-positive group was further divided into virus or bacteria subgroups. Admission day 1 blood samples were assayed for N-terminal prohormone brain natriuretic peptide, CRP, and complete blood counts.ResultsA total of 52 patients had a positive result on the array, while 20 patients had no pathogens detected. The most common bacterial pathogen detected was Haemophilus influenzae and the most common virus was rhinovirus. The pathogen-negative group had the worse outcomes with longer hospital stays (median 6.5 vs 5 days for bacteria-positive group, P=0.02) and a trend toward increased 1-year mortality (P=0.052). The bacteria-positive group had the best prognosis, whereas the virus-positive group had outcomes somewhere in between the bacteria-positive and pathogen-negative groups.ConclusionMolecular diagnostics on sputum can rapidly phenotype serious AECOPD into bacteria-, virus-, or pathogen-negative groups. The bacteria-positive group appears to have the best prognosis, while pathogen-negative group has the worst. These data suggest that AECOPD is a heterogeneous event and that accurate phenotyping of AECOPD may lead to novel management strategies that are personalized and more precise.

show abstract

“…can reduce the efficiency of DNA extraction, protocols should be optimized to the sample matrix 18 . Besides, the resistance to lysis of some bacteria, such as Gram-positive bacteria 19 , may reduce our ability to detect them, creating a bias toward more easily lysed genera. The microbial biomass in the lung is low compared to what is found in the digestive system 20 .…”

Section: Introductionmentioning

confidence: 99%

Development of a robust protocol for the characterization of the pulmonary microbiota

et al. 2021

View full text Add to dashboard Cite

The lack of methodological standardization diminishes the validity of results obtained and the conclusions drawn when studying the lung microbiota. We report the validation of a complete 16S rRNA gene amplicon sequencing workflow, from patient recruitment to bioinformatics, tailored to the constrains of the pulmonary environment. We minimize the impact of contaminants and establish negative controls to track and account for them at every step. Enzymatic and mechanical homogenization combined to commercially available extraction kits allow for a fast and reliable extraction of bacterial DNA. The DNA extraction kits have a significant impact on the bacterial composition of the controls. The bacterial signatures of extracted cancerous and healthy human tissues from 5 patients are highly distinguishable from methodological controls. Our work expands our understanding of low microbial burdened environments analysis. This article is to be a starting point towards methodological standardization and the implementation of proper sampling procedures in the study of lung microbiota.

show abstract

Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications

Cited by 9 publications

References 26 publications

Evaluation of Extraction Methods for Clinical Metagenomic Assay

Evaluation of Extraction Methods for Clinical Metagenomic Assay

<p>Phenotyping and outcomes of hospitalized COPD patients using rapid molecular diagnostics on sputum samples</p>

Development of a robust protocol for the characterization of the pulmonary microbiota

Contact Info

Product

Resources

About