Nucleic Acid Amplification via Phosphate-Free Enrichment and Sequestration
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Solution Overview
Problem
Conventional methods for detecting pathogens are time-consuming and laborious, and genetic testing methods face challenges due to inhibition of nucleic acid amplification caused by inhibitory substances in samples, leading to decreased sensitivity and false-negative results.
Innovation Solution
A nucleic acid amplification method that eliminates inhibition by forming an enrichment culture with a nutrient medium free of water-soluble phosphate ions, followed by a thermal lysis process using a lysis buffer with an organic multivalent cation-chelating reagent and a water-insoluble material to sequester interfering substances, allowing for undiluted sample use and increased sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional genetic testing methods are used, then nucleic acid amplification can be performed, but inhibition by substances in the sample reduces sensitivity and causes false-negative results
Solution Approach 1:
The patent removes water-soluble phosphate ions from the nutrient medium used in enrichment culture. This extraction of the harmful substance (phosphate ions) prevents their interference with nucleic acid amplification, thereby eliminating inhibition while maintaining detection sensitivity.
Solution Approach 2:
The patent introduces a buffer system using non-phosphate buffering agents (such as carbonate-bicarbonate, citrate, or acetate buffers) as intermediaries to maintain pH during enrichment culture without introducing inhibitory phosphate ions. These intermediary substances enable pH control while avoiding the harmful effects of phosphate.
2Reliability
If sample dilution is performed to reduce inhibition, then nucleic acid amplification can proceed, but the sensitivity of detection decreases
Solution Approach 1:
The patent converts the presence of sample substances that would normally require dilution into a benefit by using an inhibition-resistant nucleic acid amplification method. The method utilizes enzymes and conditions that are insensitive to common sample inhibitors, allowing undiluted or minimally diluted samples to be processed while maintaining both amplification success and detection sensitivity.
3Productivity
If conventional enrichment culture with phosphate buffers is used, then microorganism growth is facilitated, but phosphate ions interfere with subsequent nucleic acid amplification
Solution Approach 1:
The patent changes the chemical composition parameters of the nutrient medium by substituting phosphate buffers with alternative buffering systems (carbonate-bicarbonate, citrate, or acetate buffers). This parameter change maintains the pH-control function necessary for microorganism growth while eliminating the harmful phosphate ions that interfere with nucleic acid amplification.
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 enables rapid and sensitive detection of target microorganisms without the need for dilution or protease steps, improving the ability to detect lower concentrations of nucleic acids and reducing assay time and labor.
Implementation Method 1
The lysis buffer can comprise an organic multivalent cation-chelating reagent, wherein the organic multivalent cation-chelating reagent has a first affinity constant greater than or equal to 104.2 with respect to ferric iron
Implementation Method 2
contacting the aqueous composition with an effective amount of a water-insoluble material that sequesters a substance that interferes with a polymerase-mediated nucleic acid amplification reaction
Implementation Method 3
subjecting the aqueous composition to a thermal lysis process
Data Source
AI summary
The present disclosure provides a nucleic acid amplification method. The method comprises forming an enrichment culture by contacting a sample with a nutrient medium having a formulation that does not include a phosphate buffer component; holding the enrichment culture for a period of time at a temperature that facilitates growth of a target microorganism; after holding the enrichment culture, forming an aqueous composition by mixing a first volume of the enrichment culture with a second volume of a lysis buffer; contacting the aqueous composition with an effective amount of a water-insoluble material that sequesters a substance that interferes with a polymerase-mediated nucleic acid amplification reaction; subjecting the aqueous composition to a thermal lysis process; and, after subjecting the aqueous composition to the thermal lysis process, subjecting a portion of the aqueous composition to a nucleic acid amplification process. A composition for a lysis buffer is also disclosed.