Compositions and methods for detecting and quantifying nucleic acid sequences in blood samples
Inventors
Fischer, Jeffrey D. • Daum, Luke T. • Fischer, Gerald W.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11041216-B2
Publication Date
2021-06-22
Expiration Date
Abstract
The invention is directed to compositions and methods for rapidly detecting, amplifying, and quantitating one or more pathogen-specific nucleic acids in a biological sample, and in particular, samples obtained from patients with sepsis. The invention also provides diagnostic kits containing specific amplification primers, and labeled detection probes that specifically bind to the amplification products obtained therefrom. The invention is also directed to detecting the quantity or ratio of genomic sequences and mRNA sequences of an individual suspected of being infected with an infectious agent over time to assess the progress of the infection over time. Also disclosed are compositions and methods for the isolation and characterization of nucleic acids that are specific to one or more pathogens, such as, for example, Influenza virus, Mycobacterium tuberculosis, Plasmodium, and/or HIV from a wide variety of samples including those of biological, environmental, clinical and/or veterinary origin.
Core Innovation
The invention relates to rapidly quantifying microorganisms in a blood sample by combining the blood sample with a composition comprising a chaotrope, a detergent, a reducing agent, a chelator and a buffer, and forming a mixture for PCR-compatible processing. A portion of the mixture is combined with a PCR-ready composition that includes a heat-stable polymerase, a mix of deoxynucleotide tri phosphates, microorganism-specific PCR primers, chelating agent, osmolarity agent, albumin, at least two salts including a magnesium salt, and a buffer at a pH of about 6.5 to about 9.0, with a defined pKa relationship.
PCR amplifies the microorganism-specific sequences to form amplification products, which are quantitated to determine the quantity of microorganisms present in the blood sample. The time from collection of the sample to quantitation is less than about two days, and related approaches quantify multiple different microorganisms by contacting a biological sample with the transport/processing composition, forming reaction mixtures with PCR primer pairs specific to each microorganism, performing PCR, detecting the quantity of each amplification product, and determining the quantity of each microorganism within less than about two days.
The invention further includes using the PCR results to track infectious disease progression and to determine infection development over time. Multiple blood samples are obtained over a period of time and processed with microorganism-specific primers and a PCR-ready composition to detect the quantity of microorganisms present in each sample, and multiple biological samples can be processed to convert mRNA sequences of the infectious microorganism to DNA sequences using at least a reverse transcriptase, with PCR producing amplification products of DNA sequences of the infectious microorganism and amplification products of a genomic sequence of the individual.
Claims Coverage
The document includes four independent claims. Across these claims, the core coverage is PCR-based microorganism-specific amplification and quantification using transport/processing compositions and PCR-ready compositions with defined component sets, and applying the resulting quantities and ratios to track infection progression and determine infection development over time.
Rapid quantifying microorganisms from blood with PCR-compatible transport and PCR-ready components
combining the blood sample with a composition comprising a chaotrope, a detergent, a reducing agent, a chelator and a buffer; combining at least a portion of the mixture with a PCR-ready composition comprising a heat-stable polymerase, a mix of deoxynucleotide tri phosphates, a pair of PCR primers specific for the microorganisms, a chelating agent, an osmolarity agent, an albumin, at least two salts including a magnesium salt, and a buffer at a pH of about 6.5 to about 9.0 with a pKa within about one unit of the pH at a selected temperature; PCR amplifying to form amplification products; quantitating the amplification products to determine the quantity of microorganisms in the blood sample, wherein the time from collection to quantitation is less than about two days
Rapidly quantifying multiple microorganisms with transport composition and PCR-ready components for multiple primer pairs
contacting the biological sample with a transport composition comprising a chaotrope, a detergent, a reducing agent, a chelator and a buffer; combining the nucleic acid with pairs of PCR primer sequences specific to each different microorganism and a PCR-ready composition comprising a heat-stable polymerase, a mix of deoxynucleotide tri phosphates, a chelating agent, an osmolarity agent, an albumin, at least two salts including a magnesium salt, and a buffer at a pH of about 6.5 to about 9.0; performing PCR to form amplification products specific for each different microorganism; detecting the quantity of each amplification product; determining the quantity of each microorganism, wherein the time from collection to determining the quantity of each microorganism is less than about two days
Tracking progression of infectious disease by repeated blood sampling and microorganism-specific PCR quantification
obtaining multiple blood samples over a period of time; combining nucleic acid of each blood sample with pairs of PCR primer sequences specific to the microorganism and a PCR-ready composition comprising a heat-stable polymerase, a mix of deoxynucleotide tri phosphates, a chelating agent, an osmolarity agent, an albumin, at least two salts including a magnesium salt, and a buffer at a pH of about 6.5 to about 9.0; performing PCR to form amplification products specific for the microorganism; detecting the quantity of microorganisms present in each sample; determining the progression of the infection disease of the individual
Determining infection development by mRNA-to-DNA conversion, PCR of infectious DNA versus genomic DNA, and ratio determination
contacting each of multiple biological samples collected from the individual suspected of containing an infectious microorganism over multiple periods of time with a composition comprising a chaotrope, a detergent, a reducing agent, a chelator and a buffer; separately combining a portion of each reaction mixture with a PCR-ready composition containing at least a reverse transcriptase to convert mRNA sequences of the infectious microorganism to DNA sequences, a heat-stable polymerase, a mix of deoxynucleotide tri phosphates, a chelating agent, an osmolarity agent, an albumin, at least two salts including a magnesium salt, and a buffer at a pH of about 6.5 to about 9.0; performing PCR to produce amplification products of the DNA sequences of the infectious microorganism and amplification products of a genomic sequence of the individual; detecting the quantity and/or ratio of the amplification products in each reaction mixture; determining a ratio of amplification products from the DNA sequences and amplification products from genomic sequences to determine the development of the infection
Overall, the claims cover rapid PCR-based quantification of microorganism-specific sequences from blood or nucleic-acid-containing biological samples using a defined transport/processing composition and a PCR-ready composition with specified component categories and pH/pKa constraints, with applications to multi-pathogen quantification, tracking infection progression from repeated sampling, and determining infection development using infectious mRNA-to-DNA conversion with ratios relative to individual genomic sequences.
Stated Advantages
time from collection of the sample to quantitation is less than about two days
time from collection to determining the quantity of each microorganism is less than about two days
rapid determination of infection development by detecting quantity and/or ratio of amplification products and determining a ratio to determine development of the infection
enables determining progression of the infection disease of the individual by using quantities detected in each sample over time
supports detecting and determining the quantity of multiple different microorganisms by detecting the quantity of each amplification product
Documented Applications
tracking progression of an infectious disease of an individual by obtaining multiple blood samples over a period of time and determining progression from PCR-detected microorganism quantities
rapidly determining the development of an infection of an individual from multiple time-period biological samples by detecting quantity and/or ratio of amplification products from infectious DNA sequences versus genomic sequences
rapid PCR-based detection and quantification in blood for sepsis/bacteremia (including Mycobacterium tuberculosis bacteremia) as described in the examples
Interested in licensing this patent?