Mitochondrial multiplex real-time PCR as a source tracking method in fecal-contaminated effluents

Area of Science:

• Environmental Science
• Molecular Biology
• Water Quality Monitoring

Background:

• Surface water contamination from fecal sources poses risks to human health and ecosystems.
• Accurate identification of fecal pollution sources is crucial for effective water management and public health protection.
• Existing methods for source tracking can be labor-intensive or lack specificity.

Purpose of the Study:

• To develop and validate a multiplex real-time PCR assay for simultaneous detection of human, bovine, and swine fecal contamination in surface water.
• To assess the assay's specificity, sensitivity, and efficiency using mitochondrial DNA (mtDNA) markers.
• To evaluate the assay's performance in identifying fecal sources in complex environmental samples.

Main Methods:

• Development of species-specific primers and dual-labeled probes for human, bovine, and swine NADH dehydrogenase subunit 5 (ND5) genes.
• Application of multiplex real-time PCR to analyze fecal and effluent samples.
• Crude DNA preparation techniques for rapid sample processing.
• Validation of assay performance through linearity, amplification efficiency, and cross-reactivity testing.

Main Results:

• The multiplex real-time PCR assay demonstrated high accuracy (83% correct identification in blind tests) and specificity, with no cross-reactions observed between target species or other tested vertebrates.
• The assay showed good linearity (r2 ≥ 0.95) and amplification efficiencies (92-107%) for human, bovine, and swine mtDNA.
• The detection limit was determined to be 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent.
• Multiplexing detected the species with the highest mtDNA concentration; individual primer/probe sets improved detection of all three species in mixed effluents.

Conclusions:

• Multiplex real-time PCR targeting fecal mtDNA offers a promising, rapid, and specific method for identifying major animal and human fecal sources in surface water.
• The assay provides a valuable tool for water quality monitoring and risk assessment, complementing existing microbial source tracking techniques.
• Further optimization may be needed for detecting multiple low-concentration sources simultaneously in complex environmental matrices.