Multiplex PCR Kit for Lactococcus lactis Phage Sensitivity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for determining phage sensitivity in Lactococcus lactis starter cultures are time-consuming and expensive, particularly in large-scale screens, and do not effectively subgroup strains based on CWPS regions specific to each group.
Innovation Solution
Development of a kit and method using multiplex PCR to analyze specific gene clusters in the cwps operon, identifying unique regions that classify strains into four phage-related groups (MG-SK, IL-KF, UC-CV, and others) to determine phage sensitivity profiles, allowing for the formulation of mixed starter cultures with defined phage sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If phenotypic analysis methods (arginine hydrolysis, salt and thermal tolerance) are used for starter strain classification, then strain classification can be performed, but the process becomes time-consuming and expensive in large-scale screens
Solution Approach 1:
The patent replaces traditional phenotypic analysis methods (mechanical/chemical testing procedures) with molecular biology techniques (PCR amplification and sequencing of cwps operon regions). This substitution enables rapid, automated strain classification based on genetic markers, dramatically reducing both time and cost while maintaining or improving classification accuracy.
Solution Approach 2:
The patent uses PCR amplification to create copies of specific cwps operon regions, which serve as genetic fingerprints for strain identification. By copying and analyzing these specific genomic regions rather than performing complete phenotypic screens, the method achieves rapid classification without the time and resource costs of traditional approaches.
2Measurement precision
If traditional phenotypic analysis is applied in large-scale screens, then comprehensive strain characterization can be achieved, but the cost increases significantly
Solution Approach 1:
The patent extracts and analyzes only the specific cwps operon regions that serve as diagnostic markers for phage sensitivity, rather than performing complete phenotypic characterization. This extraction of key genetic markers enables cost-effective large-scale screening while maintaining the ability to accurately classify strains into phage sensitivity groups.
Solution Approach 2:
The patent develops a universal PCR-based methodology that can classify multiple Lactococcus lactis strains simultaneously using conserved primers targeting the cwps operon. This universal approach allows the same protocol to be applied across diverse strains and laboratories, reducing per-sample costs and enabling large-scale screens.
3Loss of information
If complete genome sequencing is performed to identify CWPS regions, then comprehensive phage sensitivity information can be obtained, but the process becomes complex and resource-intensive
Solution Approach 1:
The patent extracts only the specific cwps operon regions that contain phage sensitivity information, using targeted PCR amplification rather than complete genome sequencing. This extraction approach obtains all necessary phage sensitivity profile data while avoiding the complexity and cost of whole-genome analysis.
Solution Approach 2:
The patent segments the complex genome analysis problem into focused analysis of specific cwps operon regions. By dividing the genomic information into manageable functional segments (the cwps regions), the method simplifies both the experimental workflow and data analysis while capturing all essential phage sensitivity characteristics.
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
This approach enables efficient and cost-effective classification of Lactococcus lactis strains by their phage sensitivity, preventing fermentation failures by identifying sensitive, insensitive, and intermediate phage sensitivity strains, thereby improving cheese-making processes.
Implementation Method 1
The methods and kits of the invention are based on the discovery that a particular gene cluster/operon, designated here as cwps... The Applicant has identified three cwps-associated regions that can be used to classify strains into four major phage-related groups having varying phage-sensitivity profiles, and developed multiplex PCR for classifying strains into one of these four phage-related groups.
Data Source
Figure 1
Figure 2
Figure 2
AI summary
A kit useful for determining the phage susceptibility of one or more strains of Lactococcus lactis by means of multiplex polymerase chain reaction (PCR) is described. The kit comprises: (a) a first primer pair adapted to generate a first amplicon correlating to a region of the cwps operon that is unique to L. lactis strains MG1363 and SK11; (b) a second primer pair adapted to generate a second amplicon correlating to a region of the cwps operon that is unique to L. lactis strains IL1403 and KF147; (c) a third primer pair adapted to generate a third amplicon correlating to a region of the cwps operon that is uniqueto L. lactis strains UC509.9 and CV56; and (d) optionally, a control primer pair adapted to amplify a sequence conserved in L.lactis. The methods and kits of the invention may be employed to type strains of L. lactis into phage-sensitivity groups, and sub-type the phage-sensitivity groups according to phage sensitivity. The invention also described methods for formulation of mixed defined starter cultures using strains from different phage sensitivity groups.