Performance of bacterial and mitochondrial qPCR source tracking methods: A European multi-center study

Abstract

With the advent of molecular biology diagnostics, different quantitative PCR assays have been developed for use in Source Tracking (ST), with none of them showing 100% specificity and sensitivity. Most studies have been conducted at a regional level and mainly in fecal slurry rather than in animal wastewater. The use of a single molecular assay has most often proven to fall short in discriminating with precision the sources of fecal contamination. This work is a multicenter European ST study to compare bacterial and mitochondrial molecular assays and was set to evaluate the efficiency of nine previously described qPCR assays targeting human-, cow/ruminant-, pig-, and poultry-associated fecal contamination. The study was conducted in five European countries with seven fecal indicators and nine ST assays being evaluated in a total of 77 samples. Animal fecal slurry samples and human and non-human wastewater samples were analyzed. Fecal indicators measured by culture and qPCR were generally ubiquitous in the samples. The ST qPCR markers performed at high levels in terms of quantitative sensitivity and specificity demonstrating large geographical application. Sensitivity varied between 73% (PLBif) and 100% for the majority of the tested markers. On the other hand, specificity ranged from 53% (CWMit) and 97% (BacR). Animal-associated ST qPCR markers were generally detected in concentrations greater than those found for the respective human-associated qPCR markers, with mean concentration for the Bacteroides qPCR markers varying between 8.74 and 7.22 log₁₀ GC/10 mL for the pig and human markers, respectively. Bacteroides spp. and mitochondrial DNA qPCR markers generally presented higher Spearman's rank coefficient in the pooled fecal samples tested, particularly the human fecal markers with a coefficient of 0.79. The evaluation of the performance of Bacteroides spp., mitochondrial DNA and Bifidobacterium spp. ST qPCR markers support advanced pollution monitoring of impaired aquatic environments, aiming to elaborate strategies for target-oriented water quality management.

Keywords: Bacteroides-; Bifidobacterium DNA-targets; Fecal pollution; Human and animal wastewater; Mitochondrial-; Quantitative PCR; Source tracking.