Phage Detection Kit Using Robust qPCR Primers
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Solution Overview
Problem
Current methods for detecting and quantifying phages in dairy samples are time-consuming, labor-intensive, and often result in false negatives, making it difficult for dairy producers to take immediate corrective actions against phage-related fermentation failures, which lead to significant economic losses.
Innovation Solution
A phage DNA detection kit using conserved primers and a robust qPCR assay that allows for fast, reliable detection and quantification of phage titers in dairy samples with minimal sample treatment, capable of detecting multiple phage subgroups (c2, 936, P335, cos, pac, 5093, and 987) without the need for extensive DNA purification or pre-treatment, using a DNA polymerase such as SsoAdvanced and SYBR-related dyes for real-time results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classical overlay-method or acidification-assay is used for phage detection, then phage levels can be detected, but the detection process is time-consuming and labor-intensive
Solution Approach 1:
The patent replaces the mechanical and manual procedures of classical overlay-method and acidification-assay with a molecular biology-based qPCR system. The qPCR method uses thermal cycling and fluorescent detection to automatically quantify phage DNA, eliminating the need for manual plaque counting or pH monitoring, thereby dramatically reducing detection time while maintaining reliability
Solution Approach 2:
The patent introduces conserved primers and fluorescent dyes as intermediaries to detect phage DNA. The primers specifically bind to conserved regions of phage genomes, and the fluorescent dyes amplify the signal during qPCR, enabling rapid and sensitive detection without the time-consuming steps of traditional methods
2Reliability
If classical overlay-method or acidification-assay is used for phage detection, then phage levels can be detected, but extensive DNA purification and sample pre-treatment are required
Solution Approach 1:
The patent changes the detection parameter from phenotypic assays (plaque formation or pH change) to direct DNA detection using qPCR. This allows the use of simplified sample preparation where dairy samples are directly subjected to DNA extraction and qPCR amplification, eliminating the need for complex pre-treatment steps required by traditional methods
Solution Approach 2:
The patent extracts only the essential component (phage DNA) from the complex dairy sample matrix using simplified DNA extraction protocols. The conserved primers then specifically amplify phage DNA even in the presence of dairy inhibitors, eliminating the need for extensive purification steps
3Loss of information
If conventional phage detection methods are used, then retrospective information is obtained, but immediate corrective actions cannot be taken
Solution Approach 1:
The patent enables preliminary detection of phage DNA in dairy samples before fermentation failures occur. By using qPCR to detect phage presence and quantify titers rapidly, producers can take preventive actions such as heat treatment or sanitation procedures before phages cause fermentation problems, transforming retrospective detection into prospective prevention
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
The kit provides rapid, accurate detection and quantification of phage titers within one to two hours, enabling dairy producers to take immediate corrective actions, reducing fermentation failures and associated economic losses by offering a sensitive, specific, and efficient method for monitoring phage levels in dairy samples.
Implementation Method 1
real-time quantitative polymerase chain reaction has emerged as a method of choice to identify and quantify microbe (contaminant) species
Implementation Method 2
optimization of reaction conditions (e.g. intensity and stability of fluorophores such as SYBR-related dyes)
Data Source
AI summary
The present invention relates to a kit suitable for detection and quantification of phage DNA from a lactic acid bacteria infecting phage in a dairy sample. The invention provides a quantitative polymerase chain reaction (qPCR) kit for detection and quantification of phage DNA to from a lactic acid bacteria infecting phage in a dairy sample, said kit comprising a first primer pair and wherein said first primer pair has a robustness of a delta Cq lower than 1.0 cycle when tested in a temperature range of 55.0-70.0, preferably 55.0-68.0, more preferably 58.6-65.6 degrees Celsius and wherein said first primer pair is directed to a lactic acid bacteria infecting phage which is a lactococcal phage from the subgroup 9362, c2 or P335 or a streptococcal phage from the subgroup pac.