Sequential Lysis Protocol for Microbial Nucleic Acid Extraction
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Solution Overview
Problem
Current methods face challenges in extracting nucleic acid molecules from diverse populations of microbes in biological samples without compromising the genetic material of different microbe types, making it difficult to analyze and identify various microbial species simultaneously.
Innovation Solution
A method involving sequential lysis steps using detergent, lysozyme, and chaotropic agents, along with mechanical treatments, to effectively extract nucleic acids from a wide range of microbes, including bacteria, viruses, and fungi, allowing for metagenomics analysis and probiotic customization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a single lysis method is used to extract nucleic acid from diverse microbes, then the extraction process is simple, but the genetic material of different microbe types cannot be effectively extracted
Solution Approach 1:
The extraction process is divided into sequential lysis steps, each targeting specific microbe types: first lysis solution for gram-negative bacteria, second lysis solution with lysozyme for gram-positive bacteria, and third lysis solution with chaotropic agents for fungi and spores. This segmentation allows effective extraction from diverse microbial populations while maintaining a structured, manageable process
Solution Approach 2:
The multi-step lysis protocol serves as a universal extraction method that can handle diverse microbial types (bacteria, fungi, spores, viruses) in a single integrated workflow. Each step adds complementary functionality to broaden the scope of extractable nucleic acids while maintaining overall process coherence
2Reliability
If multiple sequential lysis steps are used to extract nucleic acid from diverse microbes, then the extraction effectiveness is improved, but the process complexity increases
Solution Approach 1:
The complex extraction challenge is broken down into manageable sequential steps, each with a specific function. The segmentation into three distinct lysis solutions with different compositions allows each step to be optimized for specific microbe types while keeping the overall process organized and systematic
Solution Approach 2:
Each lysis step is designed to act on specific microbial structures that remain after previous steps. The preliminary action of breaking down gram-negative bacteria first, then gram-positive bacteria, and finally fungi/spores creates a logical progression that simplifies the overall complex process by addressing different structural challenges in sequence
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 the successful extraction and analysis of nucleic acids from multiple microbe types in a single sample, facilitating personalized probiotic treatment and microbiome profiling, thereby improving the assessment and modulation of gut health.
Implementation Method 1
mixing the sample with a first lysis solution comprising a detergent, e.g., SDS
Implementation Method 2
a chelator, e.g., EDTA
Implementation Method 3
adding a second lysis solution having a lysozyme to the mixture
Implementation Method 4
adding a third lysis solution comprising a chaotropic agent, e.g., urea, lithium acetate, guanidine hydrochloride
Implementation Method 5
Examples of mechanical lysis include sonication
Implementation Method 6
bead mixing, and bead mill homogenization
Data Source
AI summary
Disclosed herein are methods of extracting genetic material from a diverse population of one or more types of microbes in a sample. Microbes can be prokaryotes or eukaryotes and may include bacteria, archaea, fungi, protozoa, helminths, parasites, viruses, phages, and others. Extraction may be from a single sample and subsequent identification may be through a molecular method such as qPCR, PCR, RFLP, SSCP, allele specific PCR, targeted sequencing, pull down sequencing, whole shotgun sequencing, or other methods.