Rapid extraction of nucleic acids from clinical samples for downstream applications
Inventors
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Assignees
University of California San Diego UCSD
University of California, San Diego (UCSD)The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
Publication Number
US-12359244-B2
Publication Date
2025-07-15
Expiration Date
2037-05-04
Abstract
Disclosed herein are novel methods and compositions for rapidly extracting and amplifying nucleic acids from a sample where the sample is combined with an extraction reagent comprising a reducing agent to form a mixture and incubating said mixture at ambient temperature for a period of time not exceeding 30 minutes to generate a nucleic acid extract. In certain embodiments of the method, the nucleic acid extract is subjected to a nucleic acid amplification reaction. In certain aspects, oligonucleotide primers specific for nucleic acids of Chlamydia species and/or Neisseria species are added prior to initiating the amplification reaction.
Core Innovation
The invention discloses novel methods and compositions for rapidly extracting nucleic acids from samples by combining the sample with an extraction reagent that comprises a reducing agent, such as dithiothreitol (DTT) or beta mercapto-ethanol (β-ME), and optionally a buffer. The mixture is incubated at ambient temperature for a time period not exceeding 30 minutes, generating a nucleic acid extract suitable for downstream nucleic acid amplification reactions without requiring traditional nucleic acid purification steps. This method enables rapid preparation while maintaining sample integrity for subsequent amplification.
A significant problem addressed by the invention is that current nucleic acid-based diagnostic assays require lengthy and complex nucleic acid purification procedures involving multiple steps prior to amplification. These procedures prolong diagnostic testing times, require expensive equipment and trained personnel, and complicate testing for infections such as Chlamydia trachomatis and Neisseria gonorrhoeae. The invention solves this by allowing nucleic acid extraction at ambient temperatures in a single step without purification, thereby enabling faster, simpler, and more accessible diagnostic testing.
The methods can be used with a variety of nucleic acid amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), the latter allowing amplification at a single constant temperature without the need for a thermocycler. The disclosed approach also includes using oligonucleotide primers specific for Chlamydia species, Neisseria species, or particular strains such as Lymphogranuloma venereum, enabling specific pathogen and strain identification directly from the nucleic acid extract.
Claims Coverage
The patent includes one independent claim defining a method for nucleic acid extraction and amplification. The main inventive features focus on the extraction reagent composition and conditions and the subsequent nucleic acid amplification.
Rapid ambient temperature nucleic acid extraction
Incubating a mixture comprising a sample suspected of comprising bacteria and an extraction reagent containing a reducing agent, optionally a buffer, at ambient temperature for a period not exceeding 30 minutes to generate a nucleic acid extract.
Use of reducing agents in extraction reagent
The extraction reagent comprises a reducing agent selected from dithiothreitol (DTT) or beta mercapto-ethanol (β-ME), with DTT present at 1 mM to 40 mM and β-ME at concentrations up to 40 mM.
Controlled buffer capacity and composition
The extraction reagent includes a buffer providing a buffering capacity less than that of 50 mM Tris at pH 8.5, specifically comprising Tris at 1.6 mM concentration.
Specific incubation temperature and time parameters
The ambient temperature during incubation ranges from 15° C. to 32° C., with incubation time not exceeding 30, 20, 10, 5, or even 2 minutes.
Targeting bacteria with cysteine-rich cell walls
The method is applied to samples suspected of containing bacteria, particularly those with cysteine-rich cell walls, including all Chlamydia and Neisseria species and strains.
Prompt nucleic acid amplification post-extraction
Nucleic acid amplification is initiated not later than 10 minutes, preferably not later than 5 or 2 minutes after concluding the incubation step.
Use of multiple nucleic acid amplification methods
Amplification can be performed by polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, helicase dependent amplification, or rolling circle amplification.
Inclusion of additional extraction reagent components
The extraction reagent can comprise a detergent such as Tween 20, PCR enhancers including betaine or trimethylglycine, and magnesium salts.
Use of diverse clinical sample types
Samples can be swabs from various body sites, including endocervical, vaginal, urethral, pharyngeal, conjunctival, or rectal swabs or remnant transport media thereof.
The claims cover a rapid nucleic acid extraction method using an extraction reagent with a reducing agent and optional buffer, under specific ambient incubation conditions, enabling nucleic acid amplification shortly thereafter. The method targets bacteria with cysteine-rich walls like Chlamydia and Neisseria species and supports various amplification techniques and sample types. Additives such as detergents, PCR enhancers, and magnesium salts can be included in the extraction reagent.
Stated Advantages
Allows rapid diagnostic testing for infections by eliminating lengthy nucleic acid purification steps.
Enables nucleic acid extraction and amplification to be performed at ambient room temperature without additional heating devices.
Facilitates easy sample processing where the sample collection tool (e.g., cotton swab) can be directly inserted into the extraction reagent vial.
Supports immediate or delayed nucleic acid amplification after extraction, increasing flexibility.
Compatible with multiple nucleic acid amplification methods, including isothermal amplification that do not require thermocyclers.
Allows detection of specific pathogens and strains using multiple primer sets in a single reaction.
Provides faster and easier testing for infections like Chlamydia trachomatis and Neisseria gonorrhoeae compared to existing methods.
Documented Applications
Rapid diagnostic testing for pathogenic infections by extracting nucleic acids from clinical samples such as swabs from vaginal, endocervical, urethral, rectal, pharyngeal, and conjunctival sites.
Detection and identification of bacterial pathogens including all species and strains of Chlamydia and Neisseria, with strain-specific identification such as Lymphogranuloma venereum strains.
Nucleic acid amplification for detection of genomic DNA, ribosomal RNA, or plasmid DNA from pathogens.
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