SARS-CoV-2 RT-PCR Detection Using Segmented Primer-Probe Sets

Solution Overview

Problem

There is an urgent need for methods and reagents that can detect the presence and absence of SARS-CoV-2 with high specificity and sensitivity, as existing technologies face challenges in accurately identifying the virus amidst other respiratory infections.

Innovation Solution

The development of novel nucleic acid primers and probes that target specific sequences of the SARS-CoV-2 RNA, enabling reverse transcription, amplification, and detection through RT-PCR, with sequences such as 5′-ACAACACAACAAAGGGAG-3′ and 5′-TACCGGCAGCACAAGACATCT-3′, allowing for 100% positive detection as low as 100 copies of target RNA per reaction.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional RT-PCR methods are used for SARS-CoV-2 detection, then the detection can be performed with standard reagents, but the sensitivity and specificity are insufficient to accurately distinguish SARS-CoV-2 from other coronaviruses and respiratory viruses

Engineering Contradiction:

Improvedetection specificityVSAvoidprimer and probe design complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The detection system is segmented into multiple independent components: two separate primer-probe sets targeting different regions of the SARS-CoV-2 genome (ORF1ab and N genes). Each set independently detects specific viral sequences, allowing the system to achieve high specificity by requiring concordant positive results from both targets while maintaining manageable complexity through modular design

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The primers and probes are designed with locally optimized sequences that specifically bind to unique regions of the SARS-CoV-2 genome. The forward and reverse primers for each target region are tailored to match specific local sequences, ensuring high specificity for SARS-CoV-2 detection while distinguishing it from other coronaviruses and respiratory viruses

Inventive Principle:

Principle #3Local quality

2Measurement precision

If existing detection methods are used, then the assay can be performed with standard sensitivity, but the detection limit is insufficient to detect low viral loads as low as 100 copies per reaction

Engineering Contradiction:

Improvedetection sensitivityVSAvoidamount of reagents required

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The method performs preliminary reverse transcription of the viral RNA into cDNA before the amplification step. This preliminary conversion of RNA to stable cDNA form enhances the sensitivity of subsequent PCR detection, allowing detection of very low viral loads (100 copies/reaction) by ensuring efficient conversion and preservation of the target material before amplification begins

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The detection method uses PCR amplification to create multiple copies of the viral target sequences. The forward and reverse primers facilitate exponential amplification of the cDNA templates, generating sufficient copy numbers from low initial viral loads to enable sensitive detection while maintaining efficient use of reagents through targeted amplification

Inventive Principle:

Principle #26Copying

3Adaptability or versatility

If the detection method targets multiple coronavirus types, then broader detection coverage is achieved, but the ability to specifically identify SARS-CoV-2 among other viruses is reduced

Engineering Contradiction:

Improvevirus detection coverageVSAvoidSARS-CoV-2 identification accuracy

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The detection system is segmented into multiple independent components: two separate primer-probe sets targeting different regions of the SARS-CoV-2 genome (ORF1ab and N genes). Each set independently detects specific viral sequences, allowing the system to achieve high specificity by requiring concordant positive results from both targets while maintaining manageable complexity through modular design

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The primers and probes are designed with locally optimized sequences that specifically bind to unique regions of the SARS-CoV-2 genome. The forward and reverse primers for each target region are tailored to match specific local sequences, ensuring high specificity for SARS-CoV-2 detection while distinguishing it from other coronaviruses and respiratory viruses

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS11795515B2Methods and reagents for the specific and sensitive detection of SARS-CoV-2

Publication Date: 2023.10.24 INST PASTEUR

AI summary

Methods, primers, sets of primers, probes, compositions, and kits for detecting presence or absence of SARS-CoV-2 in a sample are provided.