PNA Probes for KHV Virus Detection via Melting Curve Analysis

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for diagnosing infectious aquatic organism diseases, such as viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV/ISKNV), and Koi herpesvirus (KHV), are complex, time-consuming, and prone to false positives, necessitating improved techniques for rapid and accurate discrimination and detection.

Innovation Solution

Development of genetic markers and peptide nucleic acid (PNA) probes specific for these viruses, allowing for the discrimination and detection of VHSV, RSIV/ISKNV, and KHV through temperature-dependent melting curve analysis without the need for sequencing, using primers and PNA probes that produce distinct fluorescence intensities based on virus type.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional PCR with electrophoresis and sequencing is used for virus detection, then detection accuracy is improved, but diagnostic time and procedural complexity increase significantly

Engineering Contradiction:

Improvedetection accuracyVSAvoiddiagnostic time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The invention extracts only the essential diagnostic information (melting temperature) from the PCR product by using temperature-dependent fluorescence analysis, eliminating the need for time-consuming electrophoresis and sequencing steps while maintaining detection accuracy

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention replaces the mechanical electrophoresis separation system with a thermal analysis system that uses temperature-dependent fluorescence melting curves to distinguish viral types, significantly reducing diagnostic time and procedural complexity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If conventional PCR with cloning and sequencing is performed, then viral identification accuracy is improved, but the number of procedural steps and complexity increase

Engineering Contradiction:

Improveviral identification accuracyVSAvoidprocedural complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The invention extracts the critical diagnostic feature (melting temperature characteristic) directly from the PCR amplification step, eliminating the need for cloning and sequencing procedures while maintaining viral identification accuracy

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The PCR amplification process itself provides the diagnostic information through temperature-dependent fluorescence analysis of the amplification product, eliminating the need for separate cloning and sequencing steps

Inventive Principle:

Principle #25Self-service

3Productivity

If general PCR with fluorescent probes is used, then detection speed is improved, but measurement precision and virus type discrimination capability deteriorate

Engineering Contradiction:

Improvedetection speedVSAvoidvirus type discrimination capability

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The invention applies temperature-dependent fluorescence analysis specifically to the PCR amplification product to extract viral type-specific melting temperature characteristics, enabling both rapid detection and precise virus type discrimination simultaneously

Inventive Principle:

Principle #3Local quality

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

Enables rapid, accurate, and simple discrimination and detection of fish disease-causing viruses, reducing the complexity and time required for diagnosis and enhancing the reliability of testing.

Implementation Method 1

hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

analyzing the obtained melting curve to determine a melting temperature

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 3

controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve

Methodology Applied

Scientific EffectMelting: Melting

Data Source

PatentUS11041191B2Genetic markers for discrimination and detection of gray SPH region on Koi herprsvirus causing infectious aquatic organism diseases, and method of discriminating and detecting the virus using the same

Publication Date: 2021.06.22 NAT FISHERY PROD QUALITY MANAGEMENT SERVICE

AI summary

Genetic markers are described for discriminating or detecting viruses causing infectious aquatic organism diseases, and a method of discriminating and detecting the viruses using the same is disclosed, in which the method includes selecting and amplifying a DNA nucleotide sequence encoding a gene specific for viral hemorrhagic septicemia virus (VHSV), red sea bream iridovirus (RSIV) or infectious spleen and kidney necrosis virus (ISKNV), which is a virus causing red sea bream iridovirus disease, or Koi herpesvirus (KHV); hybridizing a peptide nucleic acid (PNA) that specifically recognizes the amplification product; controlling the temperature of the hybridization product to obtain a temperature-dependent melting curve; and discriminating the viral type or detecting whether or not fish are infected with the viral type by analyzing the obtained melting curve to determine a melting temperature.