Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation–emission matrix spectroscopy and PARAFAC

Abstract

Assays that utilize PCR offer powerful tools to detect pathogens and other microorganisms in environmental samples. However, PCR inhibitors present in nucleic acid extractions can increase a sample's limit of detection, skew calculated marker concentrations, or cause false-negative results. It would be advantageous to predict which samples contain various types and levels of PCR inhibitors, especially the humic and fulvic acids that are frequently cited as PCR inhibitors in natural water samples. This study investigated the relationships between quantitative PCR (qPCR) inhibition and the humic and fulvic content of dissolved organic matter (DOM), as well as several other measures of DOM quantity and quality, in water samples. QPCR inhibition was also compared to water quality parameters, precipitation levels, and land use adjacent to the sampling location. Results indicate that qPCR inhibition in the tested water samples was correlated to several humic substance-like, DOM components, most notably terrestrially-derived, humic-like DOM and microbially-derived, fulvic-like DOM. No correlation was found between qPCR inhibition and water quality parameters or land use, but a relationship was noted between inhibition and antecedent rainfall. This study suggests that certain fractions of humic substances are responsible for PCR inhibition from temperate, freshwater systems. PARAFAC modeling of excitation–emission matrix spectroscopy provides insight on the components of the DOM pool that impact qPCR success and may be useful in evaluating methods to remove PCR inhibitors present in samples.