Methods and compositions for determining Salmonella presence and concentration using PCR primers of varying amplification efficiencies
Inventors
Singh, Prashant • Bosilevac, Joseph M.
Assignees
United States, As Presented By Secretary Of Agriculture • Florida State University Research Foundation Inc • US Department of Agriculture USDA
Publication Number
US-12123062-B2
Publication Date
2024-10-22
Expiration Date
2042-11-11
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Abstract
Disclosed herein are methods and kits for quantifying the presence of a microorganism in a sample. Specifically, disclosed are methods for quantifying a pathogen in a sample, such as a food sample, to determine if the levels of pathogen present in the sample are within an acceptable range.
Core Innovation
The invention provides methods and kits for quantifying the presence and estimating the concentration of a microorganism, particularly pathogens such as Salmonella, in various samples including food products. The disclosed technology employs sets of PCR primers with varying amplification efficiencies to enable detection and quantification without relying on external standard curves. The method involves dividing a sample into multiple containers, each exposed to different primer sets that differ in specificity and efficiency, enabling the determination of target nucleic acid concentration based on which containers show detectable amplification.
The problem addressed by the invention is the limitation of current methods in quantifying microorganism loads rapidly and accurately. Traditional culture-based methods like most probable number (MPN) are time-consuming, requiring days to complete, and existing molecular methods depend on external standard curves that vary with sample composition and inhibitors. Conventional PCR assays mostly provide presence or absence results without estimating levels of contamination. Therefore, there is a need for rapid, reliable methods that can quantify the number of organisms present directly from samples.
The invention solves this problem by exploiting differences in primer specificity achieved through sequence mismatches, which affect amplification efficiency. This allows simultaneous detection and estimation of microorganism load, such as Salmonella, using multiplex PCR assays with carefully designed primers and probes. The assays can be performed with or without enrichment steps, employ multiple primer sets targeting conserved genes, and utilize probes specific to amplification products, thereby providing both qualitative and quantitative insights into contamination levels within shorter turnaround times than existing methods.
Claims Coverage
The patent includes three independent claims covering isolated primer pairs and nucleic acid probes for detecting amplification products.
Isolated primer pair with specified sequences
An isolated primer pair comprising nucleic acid sequences SEQ ID NOS: 1 and 2.
Primer pair with modified reverse primer for improved detection
A primer pair comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 16, differing from the first pair, enabling alternative amplification.
Nucleic acid probe with fluorochrome labels for amplification detection
A nucleic acid probe comprising SEQ ID NO: 3 linked to one or more fluorochromes selected from FITC, Texas Red, 6-FAM, dimethoxy-dichloro-fluorescein, ROX, HEX, 5-FAM, or TAMRA, for detecting amplification products produced by the respective primer pairs.
The claims collectively cover specific primer pairs for Salmonella nucleic acid amplification, including variants with differing reverse primers, and corresponding labeled probes designed for real-time detection with fluorescence, forming the basis for sensitive and specific quantification of target nucleic acids.
Stated Advantages
Rapid detection and quantification of microorganism load in samples without the need for an external standard curve.
Increased specificity and sensitivity by using multiple primer sets with varying amplification efficiencies.
Ability to detect live Salmonella cells specifically by using RNA-based assays that avoid amplification of dead cell nucleic acid.
Reduced assay complexity and turnaround time through multiplex PCR formats.
Applicability to a variety of sample types including food, clinical, and environmental samples, enabling timely and accurate decision-making for food safety.
Documented Applications
Quantifying pathogen levels in food samples such as beef, poultry, fish, shellfish, eggs, fruits, and vegetables to assess safety and compliance with regulatory standards.
Detection and estimation of live Salmonella contamination in meat processing facilities to improve public health surveillance and intervention strategies.
Use in public health and diagnostic laboratories for rapid identification and quantification of Salmonella in clinical and environmental samples.
Application in food industry settings to determine contamination levels, allowing diversion of products to appropriate processing streams or for heat treatment to mitigate foodborne illness risks.
General use in quantifying microorganisms across various biological and environmental samples, including water and clinical specimens, for diagnostic, research, and food safety purposes.
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