Background and rationale: The establishment of human gut microbiota commences initially in utero. Meconium – the first fecal material passed after birth - is used as a proxy to study fetal gut contents; however, processing meconium samples for microbiome studies presents numerous technical challenges. Meconium hosts a low biomass microbiome, is tar-like in texture, and contains high concentrations of PCR inhibitors. Previous studies report that PCR recovery of bacterial DNA from meconium can be as low as 10%. This study aimed to evaluate different DNA extraction methods to elucidate the most effective for bacterial DNA recovery and analysis from first-pass meconium.
Methods: Samples from five infants were collected under sterile conditions, divided into four 200 mg aliquots, and pre-treated with sterile Tween-80 (10%, 1 ml) to liquidise before processing. One aliquot of meconium from each infant was processed using the following extraction kits: 1) Qiagen QIAamp Stool Mini (QS); 2) Qiagen Microbiome (QM); 3) MoBio PowerSoil (PS); 4) MoBio PowerMag Microbiome (PM). Negative extraction controls were included in all cases. Bacterial DNA recovery was assessed by qPCR and endpoint PCR using universal 16S rDNA primers 357F/518R. Any PCR inhibition in samples was measured by spiking DNA eluates with 1 ng of pure Streptococcus agalactiae (GBS) DNA followed by qPCR to quantify GBS recovery.
Results: Kits QM and PM yielded the highest average DNA yields (0.266 and 0.268 ng/µl, respectively). This result is particularly interesting as kit QM isolates only prokaryotic DNA, potentially indicating that little human DNA is present in meconium. The ability of each kit to overcome PCR inhibition varied, with qPCR on GBS-spiked DNA from kits QM, QS, PS, and PM recovering 55.5%, 91.5%, 95.5% and 94.9% GBS, respectively. Endpoint-PCR confirmed that PM was the highest performing kit, with 5/5 positive samples, compared to 3/5 for QS, 4/5 for QM, and 1/5 for PS.
Conclusions: Kit PM is optimal for microbial DNA extraction from meconium.