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A loop-mediated isothermal amplification assay to detect Bacteroidales and assess risk of fecal contamination
Jiangshan Wang1, Mohsen Ranjbaran1, Aaron Ault2
1Department of Agricultural & Biological Engineering, Purdue University, West Lafayette, IN, USA; Birck Nanotechnology Center, Purdue University, West Lafayette, IN, USA.
Abstract:
Fecal contamination of fresh produce from human and animal sources is a public health concern due to the risk of foodborne illnesses. The current standard laboratory procedures for microbiological analyses usually require an enrichment step that involves several hours. Molecular techniques such as polymerase chain reaction (PCR) have been used to directly detect pathogens from the samples, however, due to the low quantity of pathogen present and small volumes used for PCR, enrichment is usually required. Additionally, the need for specialized equipment and experienced workers hinders the use of these molecular techniques for field testing. Here, we developed a rapid risk-assessment assay for fecal contamination by targeting Bacteroidales using loop-mediated isothermal amplification (LAMP). The assay allows for naked-eye observation of reactions with as few as ∼8 copies of Bacteroidales per cm2 of the surface in the field. We evaluated this assay with complex field samples as well as on-site field studies. Our on-field studies demonstrated that the Bacteroidales LAMP assay enables us to easily and quickly (<50 min) assess the risk of fecal contamination from animal operations, with a concordance of 85.3% when compared to lab-based qPCR. These results were obtained without expensive equipment (when compared to standard laboratory procedures). These assays could be used to determine site-specific risk and help the decision-making process of fresh produce growers.
Insights
A new rapid test detects fecal contamination on produce using Bacteroidales targeting loop-mediated isothermal amplification (LAMP). This field-deployable assay provides quick risk assessment for growers, complementing lab-based methods.
Area of Science:
- Food safety
- Microbiology
- Environmental science
Background:
- Fecal contamination of fresh produce poses a public health risk.
- Standard microbiological analyses and current molecular methods (like PCR) require lengthy enrichment steps and specialized equipment, hindering field application.
- Rapid, on-site detection of fecal contamination is crucial for fresh produce safety.
Purpose of the Study:
- To develop and evaluate a rapid, field-deployable assay for assessing fecal contamination on fresh produce.
- To target the Bacteroidales marker using loop-mediated isothermal amplification (LAMP) for sensitive and quick detection.
- To provide a cost-effective alternative to traditional laboratory methods for on-site risk assessment.
Main Methods:
- Development of a loop-mediated isothermal amplification (LAMP) assay targeting Bacteroidales.
- Evaluation of the assay's sensitivity, detecting as few as ~8 copies of Bacteroidales per cm2.
- Validation of the assay using complex field samples and on-site field studies, comparing results with laboratory-based quantitative PCR (qPCR).
Main Results:
- The developed Bacteroidales LAMP assay allows for naked-eye observation of results.
- The assay demonstrated high sensitivity and specificity in detecting fecal contamination.
- On-site field studies showed an 85.3% concordance with lab-based qPCR within 50 minutes, without requiring expensive equipment.
Conclusions:
- The Bacteroidales LAMP assay is a rapid, sensitive, and cost-effective tool for on-site risk assessment of fecal contamination on fresh produce.
- This assay can be easily used by fresh produce growers to make informed decisions regarding site-specific risks.
- The technology offers a practical solution for improving food safety and reducing the risk of foodborne illnesses associated with fresh produce consumption.
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