RT-PCR Reagent Cocktail for Crude Sample Inhibitor Removal

Solution Overview

Problem

Traditional reverse transcription polymerase chain reaction (RT-PCR) methods require RNA extraction from biological samples, which is labor-intensive, costly, and prone to contamination, and lacks robustness in removing inhibitors, especially in crude samples like blood.

Innovation Solution

A method and composition for RT-PCR that utilize a solution comprising a polyol, serum albumin, non-ionic surfactant, and reducing agent, such as glycerol, bovine serum albumin, Triton X-100, and dithiothreitol, allowing direct amplification of RNA from crude samples without prior extraction or purification, using a lysis and detergent neutralization step with SDS and cyclodextrin.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If RNA extraction is performed prior to RT-PCR amplification, then RT-PCR inhibitors are removed from the sample, but the process becomes labor-intensive, costly, and prone to contamination

Engineering Contradiction:

Improveremoval of RT-PCR inhibitorsVSAvoidextraction process complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent extracts and removes RT-PCR inhibitors from crude biological samples through a simplified extraction process that eliminates the need for complex RNA purification steps. The method uses selective extraction to isolate inhibitors while retaining RNA integrity, thereby simplifying the overall workflow while maintaining inhibitor removal effectiveness

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces an intermediary substance or process that facilitates the removal of inhibitors without requiring complex extraction procedures. This intermediary mechanism enables direct RT-PCR amplification from crude samples by mediating the interaction between inhibitors and the reaction system

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If RNA extraction is performed prior to RT-PCR amplification, then the amplification process is protected from inhibitors, but the time and cost of sample processing increases

Engineering Contradiction:

Improveamplification protection from inhibitorsVSAvoidsample processing time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent performs preliminary removal of RT-PCR inhibitors from crude samples before amplification, using a streamlined process that eliminates the need for time-consuming RNA extraction and purification steps. This preliminary action ensures inhibitor removal while significantly reducing overall processing time

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent skips the traditional RNA extraction and purification steps that are typically required before RT-PCR amplification. By rushing through the inhibitor removal process using a simplified method, the patent eliminates unnecessary time-consuming operations while maintaining amplification reliability

Inventive Principle:

Principle #21Skipping (Rushing through)

3Ease of operation

If direct RT-PCR amplification is performed from crude samples, then the workflow is simplified and time is saved, but inhibitor compounds interfere with amplification

Engineering Contradiction:

Improveworkflow simplicityVSAvoidamplification efficiency

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The patent extracts and removes RT-PCR inhibitors from crude biological samples through a simplified extraction process that eliminates the need for complex RNA purification steps. The method uses selective extraction to isolate inhibitors while retaining RNA integrity, thereby simplifying the overall workflow while maintaining inhibitor removal effectiveness

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces an intermediary substance or process that facilitates the removal of inhibitors without requiring complex extraction procedures. This intermediary mechanism enables direct RT-PCR amplification from crude samples by mediating the interaction between inhibitors and the reaction system

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

Enables efficient and reliable RT-PCR amplification from crude biological samples, reducing contamination risks and operational complexity, while overcoming inhibitor effects, thus improving sample processing and genetic interrogation capabilities.

Implementation Method 1

a reducing agent, which is dithiothreitol (DTT) or Tris(2-carboxyethyl) phosphine hydrochloride (TCEP)

Methodology Applied

Scientific EffectReduction: Reduction

Implementation Method 2

a non-ionic surfactant, which is Triton X-100, Brij 56 or Brij 58

Methodology Applied

Scientific EffectSurfactant: Surfactant

Implementation Method 3

a polyol, which is glycerol

Methodology Applied

Scientific EffectPolyol stabilization:

Implementation Method 4

a serum albumin, which is bovine serum albumin (BSA)

Methodology Applied

Scientific EffectProtein binding: Absorption (physical)

Data Source

PatentUS10822645B1Methods and reagents for reverse-transcription polymerase chain reaction

Publication Date: 2020.11.03 GLOBAL LIFE SCI SOLUTIONS OPERATIONS UK LTD

AI summary

The present invention provides a method of amplifying an RNA molecule in a biological sample by reverse transcription PCR (RT-PCR), wherein the RT-PCR is carried out in a solution comprising a polyol, a serum albumin, a non-ionic surfactant and a reducing agent.