qPCR Prophage Screening via ΔCt Analysis
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Solution Overview
Problem
Current methods struggle to quickly and accurately identify viable and inducible prophages from bacterial species, as non-active prophages due to genetic mutations can only be determined by functional data, and existing techniques lack efficiency in distinguishing between viable and nonviable prophage sequences.
Innovation Solution
A quantitative polymerase chain reaction (qPCR) method using consensus phage terminase DNA sequences as primers, where the difference in cycle threshold (ΔCt) values between induced and non-induced bacterial lysates indicates the presence of viable and inducible prophages, with specific ΔCt cutoffs and melting temperature ranges confirming prophage viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional functional prophage identification relying on lytic property under induction is used, then viable and inducible prophages can be identified, but the process is time-consuming and lacks efficiency
Solution Approach 1:
The patent replaces the traditional mechanical/biological assay method (plaque formation observation) with a molecular biology technique (qPCR). Instead of waiting for and observing physical plaque formation on bacterial lawns, the invention uses quantitative polymerase chain reaction to detect and measure prophage DNA copy numbers, enabling rapid and precise identification of viable and inducible prophages without the time-consuming cultural methods
Solution Approach 2:
The invention uses DNA copying through PCR amplification to detect prophage presence. By amplifying specific prophage DNA sequences and quantifying the copy numbers before and after induction, the method creates measurable copies of the genetic material that indicate viable prophage, replacing the need for time-consuming observation of physical phage particle production
2Loss of information
If genome-based identification by next generation sequencing is used, then prophage sequences can be identified, but it is difficult to distinguish viable prophages from nonviable sequences
Solution Approach 1:
The patent replaces genomic sequencing with qPCR-based quantitative detection. Instead of obtaining complete genome sequences and attempting to predict viability from sequence annotation, the invention directly measures functional activity by quantifying DNA copy number changes in response to induction, providing definitive evidence of viable prophage that sequencing alone cannot provide
Solution Approach 2:
The invention changes the detection parameter from static sequence presence (sequencing) to dynamic copy number change (qPCR). By measuring the change in DNA copy number before and after induction, the method distinguishes viable prophages that can be induced from nonviable sequences, adding a functional dimension that sequence data alone cannot provide
3Reliability
If electron microscopic determination of viral particle production is used, then viable prophages can be confirmed, but the method is complex and requires specialized equipment
Solution Approach 1:
The patent replaces electron microscopy with qPCR detection. Instead of using complex electron microscopes to visualize and count viral particles, the invention uses standard molecular biology equipment (thermocycler with fluorescence detection) to quantify prophage DNA, achieving reliable viability confirmation through a more accessible and simpler method
Solution Approach 2:
The invention uses DNA copying through PCR to create detectable signals of viable prophage presence. By amplifying specific prophage sequences and measuring copy number increases after induction, the method provides reliable viability confirmation without needing to visualize actual viral particles through complex microscopy
4Adaptability or versatility
If broad host-range prophage identification is performed using traditional methods, then functional data can be obtained, but the process is inefficient and time-consuming
Solution Approach 1:
The patent replaces traditional host range screening (requiring multiple bacterial strains and plaque assays) with qPCR-based detection. By using universal primers that target conserved prophage sequences across different bacterial species, the method can rapidly screen for viable prophages in multiple hosts simultaneously, dramatically improving productivity while maintaining the ability to determine broad host range
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 method enables rapid identification of inducible prophages, allowing for the creation of non-replicative transduction particles (NRTPs) by distinguishing viable from nonviable prophages, enhancing the efficiency of prophage detection and utilization in bacterial species.
Implementation Method 1
performing two quantitative polymerase chain reaction (qPCR) experiments with said forward and reverse primers
Data Source
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AI summary
The present invention relates to methods and compositions for performing quantitative polymerase chain reaction (qPCR) to screen for previously undiscovered inducible prophages from various bacterial strains. The present invention also relates to methods for performing qPCR to identify inducible prophages for creation of functional non-replicative transcription particles (NRTPs).