Multiplex RT-PCR Sepsis Detection Method
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Solution Overview
Problem
Current diagnostic methods for sepsis, particularly in clinical settings, face challenges such as delayed diagnosis, high costs, and limited sensitivity in detecting bacterial and fungal pathogens, leading to inappropriate antibiotic treatments and the development of multi-resistant microorganisms.
Innovation Solution
A multiplex real-time polymerase chain reaction (RT-PCR) method that discriminates between bacterial and fungal pathogens, and identifies Staphylococcus and Candida species, using a single PCR reaction with oligonucleotide primers and probes that hybridize with 16S and 18S rRNA sequences, enabling rapid and accurate detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If culture methods are used for pathogen detection, then sensitivity and accuracy are improved, but diagnosis time is significantly delayed (24-72 hours)
Solution Approach 1:
The patent replaces the mechanical culture method with a PCR-based molecular detection system. The PCR method amplifies pathogen-specific DNA sequences, enabling detection without requiring pathogen cultivation. This substitution reduces diagnosis time from 24-72 hours to several hours while maintaining high detection sensitivity through specific primer-target hybridization and exponential amplification.
2Reliability
If broad-band untargeted antibiotic therapies are applied early, then survival rates are improved, but development of multi-resistant microorganisms increases
Solution Approach 1:
The patent performs preliminary pathogen identification through PCR detection before initiating antibiotic therapy. By identifying the specific pathogen (bacterial vs. fungal, gram-positive vs. gram-negative) in advance, the system enables targeted antibiotic selection, preventing the premature use of broad-spectrum antibiotics that drive multi-resistance while ensuring appropriate treatment is started promptly.
3Adaptability or versatility
If multiplex PCR with multiple primer sets is used to detect various pathogens, then detection coverage is improved, but background signal and complexity increase
Solution Approach 1:
The patent employs universal primers that target conserved regions of bacterial 16S rRNA and fungal 18S rRNA genes. These universal primers can amplify DNA from diverse bacterial and fungal pathogens simultaneously in a single multiplex PCR reaction, providing broad detection coverage without requiring separate primer sets for each pathogen, thereby reducing reaction complexity and background signal.
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 provides a sensitive and specific means for detecting a wide range of human pathogens, reducing the need for complex nested PCR approaches and minimizing background signal, thus enabling timely and appropriate antibiotic therapy.
Implementation Method 1
using oligonucleotide primers and probes that hybridize with 16S and 18S rRNA sequences
Implementation Method 2
multiplex real-time polymerase chain reaction (RT-PCR) method
Data Source
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AI summary
The invention relates to an in vitro method for the detection and/or identification of a human pathogen and/or genetic material thereof comprising subjecting a sample comprising or suspected of comprising a human pathogen and/or genetic material thereof to a multiplex real-time polymerase chain reaction (RT-PCR), wherein said method comprises amplification of PCR products that enable discrimination between bacterial and fungal pathogens, discrimination between gram positive and gram negative bacterial pathogens, and identification of Staphylococcus and/or Candida pathogens if present in said sample. The invention also relates to the oligonucleotides used in said method and a set of oligonucleotides and a kit for carrying out the method.