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. 2023 Jun 14;11(6):531.
doi: 10.3390/toxics11060531.

Validation of a Droplet Digital PCR (ddPCR) Assay to Detect Cyanobacterial 16S rDNA in Human Lung Tissue

Rachael E Barney  1 , Guohong Huang  1 , Torrey L Gallagher  1 , Maeve Tischbein  2 , John DeWitt  3 , Rachel Martindale  3 , Ethan M P LaRochelle  1 , Gregory J Tsongalis  1 , Elijah W Stommel  2
Affiliations

Validation of a Droplet Digital PCR (ddPCR) Assay to Detect Cyanobacterial 16S rDNA in Human Lung Tissue

Rachael E Barney et al. Toxics. .

Abstract

Cyanobacteria produce a variety of secondary metabolites, including toxins that may contribute to the development of disease. Previous work was able to detect the presence of a cyanobacterial marker in human nasal and broncoalveolar lavage samples; however, it was not able to determine the quantification of the marker. To further research the relationship between cyanobacteria and human health, we validated a droplet digital polymerase chain reaction (ddPCR) assay to simultaneously detect the cyanobacterial 16S marker and a human housekeeping gene in human lung tissue samples. The ability to detect cyanobacteria in human samples will allow further research into the role cyanobacteria plays in human health and disease.

Keywords: 16S; ALS; cyanobacteria; ddPCR; human tissue.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Mean ddPCR 16S concentration (copies/mcL) of LB 2385 and LB 2390 dilutions in water, run in triplicate. LB 2385 R2 = 0.9971. LB 2390 R2 = 0.9969.
Figure 2
Figure 2
Mean ddPCR 16S concentration (copies/mcL) of LB 2385 and LB 2390 dilutions in human DNA (hDNA), run in triplicate. LB 2385 R2 = 0.9853. LB 2390 R2 = 0.9991.
Figure 3
Figure 3
Mean ddPCR RPP30 concentration (copies/mcL) of hDNA dilutions, run in triplicate. R2 = 0.9982.
Figure 4
Figure 4
16S limit of detection (LoD) for LB 2385 in hDNA is shown (1.8 copies/mcL). LB 2385 was diluted in 10 ng hDNA, with each dilution run in triplicate. Negative controls included no template controls and E. coli DNA tested at 1 pg/mcL.
See this image and copyright information in PMC

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