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Updated: Dec 31, 2025
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Published on: July 22, 2011
Pathogen Concentration Combined Solid-Phase PCR on Supercritical Angle Fluorescence Microlens Array for Multiplexed
Abstract:
Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella-spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S. typhimurium and S. enteritidis, respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.
Insights
This study presents a rapid method for detecting bloodstream infections caused by Salmonella. Combining magnetic beads and solid-phase PCR (SP-PCR) allows for ultrasensitive and quick identification of Salmonella in blood samples.
Area of Science:
- Biotechnology
- Microbiology
- Analytical Chemistry
Background:
- Bloodstream infections, particularly invasive nontyphoidal Salmonellosis, pose a significant global health and economic challenge.
- Detecting pathogens in blood is difficult due to complex sample matrices and low pathogen concentrations.
- Effective sample preparation is crucial for concentrating pathogens and removing inhibitors before detection.
Purpose of the Study:
- To develop a rapid and ultrasensitive method for detecting Salmonella enterica serovars Typhimurium and Enteritidis in blood.
- To address the challenge of pathogen detection in complex blood matrices through advanced sample preparation and detection techniques.
Main Methods:
- Combined magnetic-bead-based pathogen concentration with solid-phase PCR (SP-PCR).
- Utilized Protein G-magnetic beads immobilized with anti-Salmonella antibodies for efficient pathogen capture (>95% efficiency).
- Integrated SP-PCR on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip for enhanced detection.
Main Results:
- Achieved a limit of detection as low as 86 CFU/mL for S. Typhimurium and 94 CFU/mL for S. Enteritidis in spiked blood samples.
- Demonstrated efficient concentration of Salmonella serovars with high capturing efficiency.
- Reduced overall analysis time to under 3 hours, including sample preparation.
Conclusions:
- The integrated platform of magnetic-bead concentration and SP-PCR on a SAF microlens array enables rapid, sensitive, and accurate detection of Salmonella in blood.
- This method bypasses the need for traditional culture enrichment, significantly shortening analysis time.
- The biosensor platform shows potential for high-throughput pathogen analysis in clinical, food, and environmental samples.
