Multiplex PCR Detection of Bacteria and Fungi in Blood

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Solution Overview

Problem

Current diagnostic methods for sepsis, particularly blood cultures, are time-consuming and have low sensitivity, often requiring empirical antibiotherapy due to the challenges of detecting bacteria and fungi in blood samples, especially when antibiotics are present, which hampers effective treatment and increases mortality.

Innovation Solution

A method utilizing multiplex real-time PCR with specific primers and probes for simultaneous detection of Gram-negative and Gram-positive bacteria, as well as yeast and mold fungi, allowing for rapid and sensitive identification without the need for microbial growth, thereby overcoming the limitations of traditional blood culture methods.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If blood culture methods are used for detecting bacteria and fungi, then the detection can be performed with simple equipment and low cost, but the detection time is prolonged (up to 5 days) and sensitivity is low (only 15-20% growth rate)

Engineering Contradiction:

Improvedetection sensitivityVSAvoiddetection time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent replaces the mechanical/cultural growth-based detection system with a molecular biological system (PCR). Instead of relying on microbial growth in culture media, the invention uses polymerase chain reaction to amplify and detect microbial DNA sequences directly from blood samples, achieving rapid and sensitive detection without the time-consuming growth phase.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the detection parameter from observing microbial growth (colonies, turbidity) to detecting and amplifying DNA sequences. By targeting specific genetic markers and using PCR amplification, the method achieves high sensitivity and rapid results, fundamentally changing how detection is performed.

Inventive Principle:

Principle #35Parameter changes

2Speed

If empirical antibiotherapy is applied before blood culture, then treatment can be started immediately, but the antibiotic presence in blood inhibits microbial growth and reduces detection accuracy

Engineering Contradiction:

Improvetreatment initiation speedVSAvoiddetection accuracy

Core Design Contradiction:

SpeedVSMeasurement precision

Solution Approach 1:

The patent replaces the growth-dependent cultural method with a DNA-based molecular method. Since PCR detects genetic material directly rather than requiring live microbial growth, the presence of antibiotics does not interfere with detection. This substitution allows both rapid treatment initiation and accurate pathogen identification.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Measurement precision

If multiple separate tests are performed for different microorganisms, then each specific pathogen can be detected accurately, but the overall diagnostic process becomes complex and time-consuming

Engineering Contradiction:

Improvepathogen identification accuracyVSAvoiddiagnostic process complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent combines multiple detection capabilities into a single multiplex PCR assay. By designing primers and probes that can simultaneously detect various bacteria and fungi in one reaction mixture, the method eliminates the need for separate tests, reducing complexity while maintaining high identification accuracy for multiple pathogens.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent creates a universal diagnostic tool that can detect multiple types of microorganisms (bacteria and fungi) using a single test system. The multiplex PCR approach allows one assay to perform the work of multiple separate tests, providing broad pathogen coverage with a unified methodology.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 rapid and sensitive detection of bacteria and fungi in blood samples, reducing the need for empirical antibiotherapy and improving treatment outcomes by providing accurate identification within a shorter timeframe compared to traditional methods.

Implementation Method 1

the DNA contained in the sample of biological material is subjected to amplification in multiplex real-time PCR

Methodology Applied

Scientific EffectPCR amplification:

Data Source

PatentUS10415096B2Method for simultaneous detection of bacteria and fungi in a biological preparation by PCR, primers as well as bacteria and fungi detection kit

Publication Date: 2019.09.17 JAGIELLONIAN UNIVERSITY

AI summary

An exemplary embodiment describes a method for detection of bacteria and fungi in a sample of biological material, wherein the DNA contained in the sample of biological material is subjected to amplification in multiplex real-time PCR, with the use of primers specific for bacteria in the first stage and primers specific for fungi, and in the second stage, the resulting DNA is amplified using primers and probes differentiating fungi into a group of mold fungi and yeast fungi and bacteria into Gram-positive and Gram-negative bacteria. Another exemplary embodiment refers to oligonucleotide primers for the detection of bacteria and fungi by PCR and a kit for simultaneous detection of bacteria and fungi.