Using Droplet Digital PCR to Detect Cyanobacteria in Human Lung Tissue

OBJECTIVE AND HYPOTHESIS: Cyanobacteria produce a variety of secondary metabolites, including toxins, that are harmful to human and animal health. The objective of this study is to develop a quantitative method that can reliably detect cyanobacteria in human lung tissue. We propose that with cyanobacteria-specific 16S rDNA primers, droplet digital PCR (ddPCR) can provide a reliable and sensitive method to detect Cyanobacteria in human tissue. METHODS: Two cyanobacteria strains were used in this validation: a toxin-producing strain, Microcystis aeruginosa (LB 2385), and a non-toxin-producing, strain Synechococcus sp. (LB 2390). Escherichia coli served as a negative bacterial control. DNA was extracted from cultures and frozen, human lung tissue with DNeasy Blood & Tissue kit (QIAGEN) and quantified using the dsDNA High-Sensitivity Assay (Invitrogen) on a Qubit 3.0 fluorometer. The ddPCR reactions consisted of FAM labelled cyanobacterial 16S rDNA target primer/probe and HEX labelled RPP30 reference primer/probe (Bio-RAD). Oil droplets were generated using the Bio-Rad Automated Droplet Generator and the emulsified PCRs were run in a 96-well plate on a C1000 Touch™ Thermal Cycler. The plates were read on a Bio-Rad QX200 droplet reader and the data analyzed using the QuantaSoft v1.7.4 software. The Limit of Detection (LOD) was found by initially diluting LB 2385 and LB 2390 to 4 ng/mcL in nuclease-free water, followed by serial 2x dilutions until it was below the detection limit by ddPCR. This was repeated using normal human lung tissue instead of nuclease-free water. RESULTS: The ddPCR assay developed in this study showed high specificity and sensitivity in detecting both strains of cyanobacteria. Under both conditions tested (spiking in water vs 10 ng human DNA (hDNA)), LB 2385 DNA could be reliably detected at a concentration of 61 fg/mcL, whereas LB 2390 DNA could be reliably detected at a concentration of 31 fg/mcL. This corresponds to 1-2 copies/mcL of cyanobacterial DNA in the original samples. In the presence of hDNA, the sensitivity for cyanobacterial DNA decreased as the concentration of hDNA increased (2-30 ng/mcL), possibly due to a physical barrier or competition between the primer/probes. In the hDNA condition, RPP30 could be reliably detected at 2-3 copies/mcL with a concentration of ~60 pg/mcL. RPP30 is valuable for normalizing sample input, reducing biases caused by DNA quantification variations and pipetting errors. A strong linear correlation between sample input and the number of positive droplets (i.e., the concentration detected by ddPCR) was observed for both cyanobacteria 16S rDNA and RPP30 DNA targets. This assay also demonstrated high intra- and inter-assay reproducibility. CONCLUSIONS: A highly specific and sensitive ddPCR assay was developed to detect the presence of cyanobacteria in human lung tissue. The ability to detect cyanobacteria in human samples will allow further research into the role cyanobacteria plays in human health and disease. © FASEB.