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Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis.
Phage. 2020 Jun 01; 1(2):100-108.P

Abstract

Background:

There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and

Methods:

In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum.

Results:

Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies.

Conclusion:

These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF.

Authors+Show Affiliations

Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, California, USA.Division of Biological Sciences, University of Montana, Missoula, Montana, USA.Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, California, USA.Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, California, USA.Harbers Bik LLC, San Francisco, California, USA.Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, Stanford, California, USA.Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, California, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32626852

Citation

Burgener, Elizabeth B., et al. "Methods for Extraction and Detection of Pf Bacteriophage DNA From the Sputum of Patients With Cystic Fibrosis." PHAGE (New Rochelle, N.Y.), vol. 1, no. 2, 2020, pp. 100-108.
Burgener EB, Secor PR, Tracy MC, et al. Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis. Phage. 2020;1(2):100-108.
Burgener, E. B., Secor, P. R., Tracy, M. C., Sweere, J. M., Bik, E. M., Milla, C. E., & Bollyky, P. L. (2020). Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis. PHAGE (New Rochelle, N.Y.), 1(2), 100-108. https://doi.org/10.1089/phage.2020.0003
Burgener EB, et al. Methods for Extraction and Detection of Pf Bacteriophage DNA From the Sputum of Patients With Cystic Fibrosis. Phage. 2020 Jun 1;1(2):100-108. PubMed PMID: 32626852.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Methods for Extraction and Detection of Pf Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis. AU - Burgener,Elizabeth B, AU - Secor,Patrick R, AU - Tracy,Michael C, AU - Sweere,Johanna M, AU - Bik,Elisabeth M, AU - Milla,Carlos E, AU - Bollyky,Paul L, Y1 - 2020/06/16/ PY - 2021/07/01/pmc-release PY - 2020/7/7/entrez PY - 2020/7/7/pubmed PY - 2020/7/7/medline KW - DNA extraction KW - bacteriophage KW - cystic fibrosis KW - sputum SP - 100 EP - 108 JF - PHAGE (New Rochelle, N.Y.) JO - Phage VL - 1 IS - 2 N2 - Background: There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and Methods: In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum. Results: Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies. Conclusion: These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF. SN - 2641-6549 UR - https://www.unboundmedicine.com/medline/citation/32626852/Methods_for_Extraction_and_Detection_of_Pf_Bacteriophage_DNA_from_the_Sputum_of_Patients_with_Cystic_Fibrosis DB - PRIME DP - Unbound Medicine ER -
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