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Modified PCR protocol to increase sensitivity for determination of bacterial community composition.
Williamson, Kayla M; Wagner, Brandie D; Robertson, Charles E; Stevens, Mark J; Sontag, Marci K; Mourani, Peter M; Harris, J Kirk.
Afiliação
  • Williamson KM; Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado School of Medicine, 13001 17th Place, Mail Stop B119, Aurora, CO, 80045, USA.
  • Wagner BD; Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado School of Medicine, 13001 17th Place, Mail Stop B119, Aurora, CO, 80045, USA.
  • Robertson CE; Section of Critical Care, Department of Pediatrics, University of Colorado School of Medicine and Children's Hospital Colorado, 13123 E. 16th Ave. Box B395, Aurora, CO, 80045, USA.
  • Stevens MJ; Division of Infectious Diseases, School of Medicine, University of Colorado, 12700 East 19th Avenue, Mail Stop B168, Aurora, CO, 80045, USA.
  • Sontag MK; Section of Critical Care, Department of Pediatrics, University of Colorado School of Medicine and Children's Hospital Colorado, 13123 E. 16th Ave. Box B395, Aurora, CO, 80045, USA.
  • Mourani PM; Department of Epidemiology, Colorado School of Public Health, University of Colorado School of Medicine, 13001 17th Place, Mail Stop B119, Aurora, CO, 80045, USA.
  • Harris JK; Section of Critical Care, Department of Pediatrics, University of Colorado School of Medicine and Children's Hospital Colorado, 13123 E. 16th Ave. Box B395, Aurora, CO, 80045, USA.
Microbiome ; 9(1): 90, 2021 04 13.
Article em En | MEDLINE | ID: mdl-33849648
BACKGROUND: The objective of this project was to increase the sensitivity of sequence-based bacterial community determination without impacting community composition or interfering with cluster formation during sequencing. Two PCR protocols (standard and modified) were examined in airway samples where we observed a large range in bacterial load (3.1-6.2 log10 16S rRNA gene copies/reaction). Tracheal aspirate (TA) samples (n = 99) were collected from sixteen children requiring mechanical ventilation at a single center. DNA was extracted, and total bacterial load (TBL) was assessed using qPCR. Amplification of 16S rRNA was attempted with both protocols in all samples. RESULTS: PCR product was observed using both protocols in 52 samples and in 24 additional samples only with the modified protocol. TBL, diversity metrics, and prominent taxa were compared for samples in three groups based on success of the two protocols (successful with both, success with modified only, unsuccessful for both). TBL differed significantly across the three groups (p<0.001). Specifically, the modified protocol allowed amplification from samples with intermediate TBL. Shannon diversity was similar between the two protocols, and Morisita-Horn beta diversity index showed high agreement between the two protocols within samples (median value 0.9997, range 0.9947 to 1). We show that both protocols identify similar communities, and the technical variability of both protocols was very low. The use of limited PCR cycles was a key feature to limit impact of background by exclusion of 24% of samples with no evidence of bacterial DNA present in the sample. CONCLUSION: The modified amplification protocol represents a viable approach that increased sensitivity of bacterial community analysis, which is important for study of the human airway microbiome where bacterial load is highly variable. Video abstract.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Bactérias / Microbiota Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Child / Humans Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Bactérias / Microbiota Tipo de estudo: Diagnostic_studies / Prognostic_studies Limite: Child / Humans Idioma: En Ano de publicação: 2021 Tipo de documento: Article