Multiplex PCR Assay for Bordetella Species Differentiation

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Solution Overview

Problem

Current diagnostic methods for detecting Bordetella pertussis, B. parapertussis, and B. holmesii are either insensitive, time-consuming, or prone to false diagnoses, particularly in complex clinical samples, necessitating a rapid and specific detection method to accurately diagnose pertussis-like diseases.

Innovation Solution

A composition and kit utilizing specific nucleotide sequences and primers for real-time PCR, allowing for the selective and multiplex detection and differentiation of B. pertussis, B. holmesii, and B. parapertussis, using primers and probes designed to amplify unique regions of the Bordetella species, enabling rapid and accurate diagnosis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If culture method is used to detect B. pertussis, then specificity is improved (100% specific), but sensitivity deteriorates (12-60% sensitive) and time consumption increases (5-10 days incubation)

Engineering Contradiction:

ImprovespecificityVSAvoidsensitivity

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent replaces the mechanical culture system with a molecular biology system (PCR). Specifically, it uses real-time PCR with species-specific primers and probes to detect Bordetella DNA directly from clinical samples, eliminating the need for prolonged cultural incubation while achieving both high sensitivity and high specificity through molecular identification.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the detection parameter from observing bacterial growth (culture) to detecting specific DNA sequences (PCR). By targeting species-specific genetic markers such as IS481 for B. pertussis and IS1001 for B. parapertussis, the method achieves rapid identification within hours rather than days, while maintaining species-specific accuracy.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If standard real-time PCR assays are used to detect Bordetella species, then detection speed is improved (2-24 h), but diagnostic accuracy deteriorates due to false positives from cross-reactivity with other Bordetella species

Engineering Contradiction:

Improvedetection speedVSAvoiddiagnostic accuracy

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent segments the detection process into species-specific reactions. It uses distinct primer and probe sets for each Bordetella species: IS481-targeting reagents for B. pertussis, IS1001-targeting reagents for B. parapertussis, and species-specific probes for B. holmesii. This segmentation allows simultaneous detection of multiple species in a single multiplex reaction, eliminating cross-reactivity false positives while maintaining rapid detection.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces species-specific DNA probes as intermediaries that selectively bind to target sequences. These probes act as mediators between the PCR amplification process and the detection system, ensuring that only the intended Bordetella species generates a positive signal. The probes contain species-specific recognition sequences that prevent cross-reactivity with non-target species.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 solution provides a rapid, sensitive, and specific assay for detecting Bordetella species, reducing false diagnoses and improving diagnostic accuracy in clinical and laboratory settings, capable of distinguishing between species and co-infections.

Implementation Method 1

Methodology Applied

Scientific EffectPolymerase Chain Reaction (PCR):

Data Source

PatentEP3006565B1Selective detection of bordetella parapertussis

Publication Date: 2020.03.25 THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HEALTH & HUMAN SERVICES

Figure 1A~1B
Figure 2

AI summary

A process for detecting Bordetella spp. nucleic acid in a biological sample includes producing an amplification product(s) by amplifying one or more Bordetella spp. in a multiplex single chamber PCR assay, and measuring said amplification product(s) to detect or distinguish Bordetella spp. in the biological sample. Also provided are reagents and methods for detecting and distinguishing Bordetella spp. from each other and other bacteria or viruses. A kit is provided for detecting and quantifying one or more Bordetella spp. in a biological sample.