Multiplex rRT-PCR Assay for SARS-CoV-2 Detection

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Solution Overview

Problem

Current nucleic acid amplification tests for SARS-CoV-2 detection lack sensitivity, specificity, and speed, leading to inefficiencies in diagnosing the virus and its variants.

Innovation Solution

A multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) method that simultaneously amplifies and detects specific segments of the human RP gene and SARS-CoV-2 RdRP and E or N2 genes using specially designed primers and probes, allowing for faster and more accurate detection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional nucleic acid amplification tests are used for SARS-CoV-2 detection, then the detection can be performed, but the sensitivity, specificity, and speed are insufficient

Engineering Contradiction:

Improvedetection sensitivity and specificityVSAvoiddetection speed

Core Design Contradiction:

ReliabilityVSSpeed

Solution Approach 1:

The patent combines multiple detection targets (viral N gene, E gene, and human RNase P gene) into a single multiplex PCR reaction system. By merging these detection functions into one assay, the system achieves high sensitivity and specificity through multi-target validation while maintaining fast detection speed comparable to single-target tests, thus resolving the contradiction between reliability and speed.

Inventive Principle:

Principle #5Merging (Combining)

2Reliability

If multiple separate PCR tests are performed to ensure accurate detection, then sensitivity and specificity improve, but the detection time and complexity increase

Engineering Contradiction:

Improvedetection accuracyVSAvoidtotal detection time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The invention merges multiple PCR detection reactions into a single multiplex reaction tube by carefully designing primers and probes that can simultaneously amplify and detect different targets (viral N gene, E gene, and human RNase P gene) without interfering with each other. This allows accurate multi-target detection to be completed in one reaction run rather than requiring multiple separate tests, thereby maintaining high detection accuracy while significantly reducing the total detection time.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The multiplex PCR system serves multiple detection functions simultaneously within a single assay platform. The same reaction system can detect the presence of viral RNA through multiple viral gene targets while also monitoring the quality of the sample through the human RNase P gene amplification, making the system universally applicable for both viral detection and sample quality control in a single operation.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Productivity

If a multiplex PCR system is designed to detect multiple viral genes simultaneously, then detection efficiency improves, but the risk of false positives and cross-contamination increases

Engineering Contradiction:

Improvedetection efficiencyVSAvoidfalse positive rate

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent combines multiple detection functions in a single multiplex PCR system with carefully optimized primer and probe concentrations. By merging the detection of viral N gene, E gene, and human RNase P gene into one reaction with balanced reagent ratios, the system achieves high detection efficiency while maintaining reliability through the use of multiple independent detection targets that reduce false positives.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The multiplex system incorporates the human RNase P gene as an internal control that provides feedback on the quality and integrity of the extracted RNA sample. If the RNase P gene does not amplify properly, it indicates sample degradation or extraction issues, allowing the system to flag potential false results before viral gene detection, thus reducing false positive rates while maintaining high productivity.

Inventive Principle:

Principle #23Feedback

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 highly sensitive and efficient means to detect SARS-CoV-2 RNA, reducing reaction time and effort while maintaining high specificity, enabling early and accurate diagnosis of the virus and its variants.

Implementation Method 1

contacting cDNA produced from SARS-CoV-2 RNA with primers that amplify human RP, viral RdRP, and viral E or N2 genes, dNTPs, and a DNA polymerase under conditions suitable for amplification of the cDNA

Methodology Applied

Scientific EffectPolymerase chain reaction:

Implementation Method 2

contacting the amplified cDNA with fluorescent detection probes that bind to amplified human RP, viral RdRP, and viral E or N2 genes

Methodology Applied

Scientific EffectNucleic acid hybridization:

Implementation Method 3

cDNA produced from SARS-CoV-2 RNA

Methodology Applied

Scientific EffectReverse transcription:

Data Source

PatentUS20220195540A1Multiplex real-time RT-PCR method for the diagnosis of SARS-COV-2 by targeting viral e, rdrp and human RP genes or viral n2, rdrp and human RP genes

Publication Date: 2022.06.23 IMAM ABDULRAHMAN BIN FAISAL UNIV

AI summary

A method for detecting SARS-CoV-2 RNA or cDNA in a sample comprising real-time reverse transcription polymerase chain reaction that specifically amplifies and detects nucleic acid sequences amplified by primers to human RP gene, and SARS-CoV-2 RdRP and E, or SARS-CoV-2 RdRP and N2 genes. Specific primers and fluorescent probes that amplify and detect specific segments of human RP gene and SARS-CoV-2 RdRP and E, or SARS-CoV-2 RdRP and N2 genes with high sensitivity and efficiency compared to conventional methods.