Optimizing Lysis and Extraction Workflows for Enrichment-Free qPCR Detection of Salmonella enterica in Poultry Matrices

TL;DR

The study improves qPCR detection of Salmonella in poultry by optimizing lysis and extraction methods without needing enrichment.

Contribution

A systematic optimization of lysis and DNA extraction workflows for direct qPCR detection of Salmonella in poultry matrices.

Findings

Lysis with 20 µL Proteinase K and 400 µL PrepMan™ for 20 min yielded the lowest and most consistent Cq values.

Broth-derived inocula produced earlier and more reproducible Cq values than colony-derived preparations.

MNP processing increased Cq values compared to non-MNP workflows in both matrices.

Abstract

Salmonella remains a leading cause of foodborne illness worldwide, with poultry products representing a major source of human exposure, underscoring the need for rapid and reliable detection methods. Although qPCR offers sensitive and timely pathogen detection, assay performance is highly dependent on sample preparation efficiency and nucleic acid purity, particularly in complex food matrices. In this study, we systematically optimized the sample preparation workflow of a SYBR Green based qPCR assay for enrichment-free detection of Salmonella enterica in poultry. Multiple lysis chemistries, incubation times, DNA extraction methods, centrifugation strategies, inoculum sources, and magnetic nanoparticle (MNP) assisted workflows were evaluated using phosphate-buffered saline and chicken rinsate matrices. Among the conditions tested, lysis with 20 µL Proteinase K and 400 µL PrepMan™ for 20 min produced the lowest and most consistent Cq values. Although Promega Wizard® produced slightly lower mean Cq values than PrepMan™, statistical analysis showed no significant differences between extraction methods or centrifugation protocols, indicating comparable overall performance. Broth-derived inocula yielded earlier and more reproducible Cq values than colony-derived preparations. In contrast, inclusion of MNP processing resulted in higher Cq values in both matrices compared to the non-MNP workflow. Overall, these findings demonstrate that optimized lysis, extraction, and centrifugation workflows enhances the consistency and analytical reliability of direct qPCR detection of Salmonella in poultry matrices, supporting laboratory-based rapid detection applications.

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