Services Methods
FAQs
What is 16S? 16S is a region of bacterial DNA (genetic material) that is used to identify the taxonomic group (genus, species, etc.) to which a bacterium belongs.
What is ITS? Likewise, ITS is a region of fungal DNA that is used to identify the taxonomic group to which a fungus belongs.
What is qPCR? qPCR is a mode of analyzing genetic material or DNA, such that we learn about whether certain microorganisms are present but also, how relatively abundant they are.
Technical Methods
DNA Extraction: For each sample, a 0.250 g subsample was collected from the material received. Samples experienced total microbial community DNA extraction using the Qiagen DNeasy PowerSoil Pro DNA Extraction Kit paired with Qiagen’s QiaCube Connect DNA extraction robot (Qiagen, Hilden, Germany).
DNA Sample Quantification: 2.0 μL of each extracted DNA sample was quantified in units of nanogram per microliter (ng / μL) using a Qubit Flex Fluorometer. Sample gDNA values were normalized to 2.00 ng / µL prior to qPCR amplification, to show a relative quantification of each sample to one another.
Continuµm Establishment qPCR: Bacterial abundances were quantified using qPCR, targeting regions specific to each of the four bacterial species in Impello’s Continuµm product. A fivefold dilution series of each bacterium’s genomic DNA (gDNA) was used to generate a standard curve for quantification, with gDNA standards prepared at concentrations of 1000, 100, 10, 1, and 0.1 pg/μL. The Ct values from unknown samples were plotted onto the standard curve’s line of best fit for quantification.
Fusarium spp. qPCR: The abundances of the members within the fungal genus Fusarium were quantified using another qPCR approach. A standard curve was generated using Fusarium oxysporum gDNA at 10 x dilutions of 1000, 100, 10, 1, and 0.1 pg/µL, with molecular water as a negative control (0 pg/µL). Each biological DNA sample, extracted from rhizosphere samples, was fractionated into three technical replicates. The Ct values from unknown samples were plotted onto the standard curve’s line of best fit for quantification.
Fungal and Bacterial abundance qPCR: Bacterial and fungal abundances were quantified using (qPCR) targeting 16S or ITS amplicons. For each assay, a fivefold dilution series of Pseudomonas putida or Aspergillus niger genomic DNA (gDNA) was used to generate a standard curve. Standards for each 16S or ITS assay were prepared at the following concentrations: 1000 pg/µL, 100 pg/μL, 10 pg/μL, 1 pg/μL and 0.1 pg/μL. For each assay, quantification of bacterial and fungal populations from the unknown samples was conducted by plotting the qPCR-resulting Ct values from unknown samples onto a line of best fit from the standard curve results.