Multiplex PCR Detection of TAB in Foodstuffs
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Solution Overview
Problem
Traditional methods for detecting foodstuff-spoiling Thermophilic Acidophilic Bacteria (TAB), such as Alicyclobacillus species, are time-consuming and inefficient, leading to significant economic losses in the beverage industry due to their heat-resistant and acid-tolerant nature, which allows them to survive pasteurization and cause spoilage through guaiacol production.
Innovation Solution
The use of Polymerase Chain Reaction (PCR) techniques with specific oligonucleotide primers targeting nucleic acids of guaiacol-producing microorganisms and Alicyclobacillus acidoterrestris, allowing for rapid and accurate detection of TAB contamination through multiplex PCR amplification and analysis of PCR products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If traditional microbiology methods are used to detect TAB, then detection accuracy is achieved, but detection time is excessively long (at least five days)
Solution Approach 1:
The patent replaces traditional mechanical microbiology methods (culture-based detection requiring incubation and manual observation) with PCR-based molecular detection. This substitution enables amplification of target DNA sequences specific to TAB, allowing detection within hours rather than days, thus dramatically reducing detection time while maintaining high accuracy.
Solution Approach 2:
The patent employs preliminary enrichment culture steps before PCR detection to concentrate TAB DNA from the food sample. This preliminary action ensures sufficient target DNA is available for rapid amplification and detection, enabling the overall process to be completed in less than five days while maintaining sensitivity.
2Reliability
If traditional microbiology methods are used, then comprehensive detection is possible, but the process is too slow for rapid quality control
Solution Approach 1:
The patent substitutes traditional slow microbiological cultivation and identification methods with PCR amplification followed by sequencing or probe-based detection. This molecular approach maintains comprehensive detection capability while reducing the timeline from weeks to hours, enabling rapid quality control without sacrificing reliability.
Solution Approach 2:
The patent changes the detection parameter from observing phenotypic characteristics (growth patterns, colony morphology) to detecting genotypic characteristics (specific DNA sequences). This parameter change allows for much faster identification since DNA amplification and analysis can be performed rapidly compared to waiting for microbial growth and observation.
3Productivity
If PCR-based detection is implemented, then detection speed is dramatically improved, but method complexity increases
Solution Approach 1:
The patent extracts and isolates specific DNA sequences from the complex food matrix that are unique to TAB. By focusing detection on these specific genetic markers rather than attempting to detect all microorganisms, the method achieves high efficiency while managing complexity through targeted rather than universal detection.
Solution Approach 2:
The patent uses specific DNA primers and probes as intermediaries to bridge the gap between the complex food sample and the detection system. These intermediaries selectively bind to TAB-specific DNA sequences, enabling efficient detection while simplifying the overall process by filtering out non-target DNA through specific molecular recognition.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient and accurate genus- and species-level detection of Alicyclobacillus, including identification of guaiacol-producing strains, within a significantly reduced timeframe, typically less than five days.
Implementation Method 1
contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprising: a first collection of primers targeted for nucleic acids of a guaiacol-producing microorganism; and a second collection of primers targeted for nucleic acids of Alicyclobacillus acidoterrestris
Implementation Method 2
amplifying DNA in the sample with the said set of oligonucleotides under a multiplex polymerase chain reaction (PCR)
Data Source
AI summary
The present disclosure provides compositions, methods, and kits for detecting guaiacol-producing microorganisms, in particular, Thermophilic Acidophilic Bacteria (TAB) in foodstuff. In one example, a method comprises: (a) contacting a set of oligonucleotides with the sample, wherein the set of oligonucleotides comprising: a first collection of primers targeted for nucleic acids of a guaiacol-producing microorganism; and a second collection of primers targeted for nucleic acids of Alicyclobacillus acidoterrestris; (b) amplifying DNA in the sample with the said set of oligonucleotides under a multiplex polymerase chain reaction (PCR); and (c) determining the presence of PCR products of step (b), wherein the presence of a PCR product in the sample is indicative of contamination of the sample by both guaiacol-producing microorganism and Alicyclobacillus acidoterrestris.