Methods for species-level resolution of microorganisms
Inventors
KEMP, Ryan • Weinstein, Mike • Tang, Shuiquan
Assignees
Publication Number
US-11807909-B1
Publication Date
2023-11-07
Expiration Date
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Abstract
Methods and devices are provided for the identification and/or quantification of microbes. In particular, the methods comprise: (a) extracting DNA from a microbial sample; (b) performing 16S ribosomal RNA gene-targeted sequencing to obtain DNA sequences; and (c) analyzing the DNA sequences to identify the species of the one or more microbes.
Core Innovation
Methods and devices are provided for the identification and/or quantification of microbes. In particular, the methods comprise: (a) extracting DNA from a microbial sample; (b) performing 16S ribosomal RNA gene-targeted sequencing to obtain DNA sequences; and (c) analyzing the DNA sequences to identify the species of the one or more microbes.
In many microbiology-related applications (e.g., infectious disease diagnosis and food safety testing), the need to determine the identity and quantity of the microbes is a common requirement. Problems with culture-based microbial detection have been widely recognized including limited coverage as many microbes are not culturable, slow as many culturable microbes take days or even weeks to grow colonies, difficult as many anaerobes are very difficult to cultivate, and multiple tests as it takes several tests to cover potential pathogens; qPCR can be relatively fast and low cost but can only cover limited targets.
The present disclosure provides an NGS-based 16S ribosomal RNA gene-targeted sequencing approach with high sensitivity and dramatically larger coverage compared to culture-based or qPCR methods, providing species-level resolution of microorganisms and a turnaround time that may be about 2 days; certain aspects state the method does not comprise microbial culture or array analysis.
Claims Coverage
One independent claim was identified. Four main inventive features were extracted from the independent claim.
Extraction of DNA from a microbial sample
extracting DNA from a microbial sample
16S ribosomal RNA gene-targeted sequencing
performing 16S ribosomal RNA gene-targeted sequencing to obtain DNA sequences
Analyzing DNA sequences to identify species
analyzing the DNA sequences to identify the species of the one or more microbes
Primer pool for sequencing
wherein the sequencing comprises using a pool of V1-V2 primers comprising SEQ ID NOs: 1-11 and/or a pool of V3-V4 primers comprising SEQ ID NOs: 12-19
The independent claim covers a workflow comprising DNA extraction, 16S rRNA gene-targeted sequencing, and sequence analysis to identify species, with explicit recitation of primer pools for V1-V2 and/or V3-V4 (SEQ ID NOs: 1-11 and 12-19).
Stated Advantages
High sensitivity.
Dramatically larger coverage compared to culture-based or qPCR methods.
Provides species-level resolution of microorganisms as compared to previous methods which can be limited to genus-level resolution.
Turnaround time may be about 2 days; the method is stated as performable in less than 3 days and in 2 days in certain aspects.
The method does not comprise microbial culture or array analysis in certain aspects.
New 16S primers were introduced to provide coverage for microbes that are otherwise missed in common 16S primer sets.
A bioinformatics pipeline (use of FIGARO prior to DADA2 and a unique taxonomy assignment function with a well-curated 16S gene database) enables species-level resolution for bacteria identification.
Documented Applications
Infectious disease diagnosis.
Food safety testing.
Microbiome or metagenome analyses.
Study the natural diversity of bacterial species and resolve unclassified isolates into novel species or strains.
Define regional and global epidemiology of bacterial species, study the epidemiology of infectious diseases, differentiate pathogenic organisms, and study endemic or epidemic strains.
Identify bacterial contamination sources (e.g., hospital-acquired/nosocomial, foodborne, animal/zoonosis, environmental), link patient acquisition of a bacterial infection, and track patient-to-patient transmission during infectious disease outbreaks.
Inform decisions regarding infection prevention and control measures in health care facilities and inform decisions regarding public health response to outbreaks.
Analysis of human samples and environmental samples including water, biofilms, soil, air, or host-derived samples (e.g., body fluids, saliva, urine, fecal, surface swab, root, leaf or bark samples).
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