Compositions and methods for detecting and quantifying nucleic acid sequences in blood samples
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.
1. A method of rapidly quantifying microorganisms in a blood sample comprising:
combining the blood sample with a composition to form a mixture, wherein the composition comprises 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 to form a reaction mixture containing microorganism-specific sequences, wherein the PCR-ready composition comprises as components a heat-stable polymerase; a mix of deoxynucleotide tri phosphates; a pair of PCR primers configured to amplify by PCR a nucleic acid sequence that is specific for the microorganisms; a chelating agent; an osmolarity agent; an albumin; at least two salts at least one of which is a magnesium salt; and a buffer present at a pH of about 6.5 to about 9.0, wherein the pKa of the composition is within about one unit of the pH at a selected temperature, wherein the components are combined with nuclease-free water;
PCR amplifying the microorganism-specific sequences of the reaction mixture to form amplification products; and
quantitating the amplification products to determine the quantity of microorganisms present in the blood sample, wherein the time from collection of the sample to quantitation is less than about two days.
2. The method of claim 1 , wherein the heat-stable polymerase is a Taq polymerase, a high fidelity polymerase, a Pfu polymerase, a hot start polymerase, or a next gen polymerase.
3. The method of claim 1 , wherein composition further comprises a dye.
4. The method of claim 3 , wherein the dye is selected from the group consisting of fluorescein, 5-carboxy-X-rhodamine and ROX.
5. The method of claim 1 , wherein the pH of the composition is from about 6.5 to about 7.5 and the pKa is within 0.5 of the pH of the buffer at ambient temperature.
6. The method of claim 5 , wherein the pKa of the composition is within 0.2 of the pH of the buffer at ambient temperature.
7. The method of claim 1 , wherein the PCR-ready composition has a pH from about 6.5 to 7.0.
8. The method of claim 7 , wherein the pair of PCR primers are each from about 18 to 35 nucleotides in length.
9. The method of claim 1 , wherein the microorganisms are bacteria, virus, fungi, parasites or combinations thereof.
10. The method of claim 9 , wherein the bacteria are MTB, the virus is HIV, or the parasites is a Plasmodium species and/or Plasmodium falciparum.
11. The method of claim 1 , further comprising a control nucleic acid present in the PCR-ready composition at a concentration of about 1 fg to about 1 ng.
12. The method of claim 1 , wherein the time from collection of the sample to quantitation is less than about one day.
13. The method of claim 1 , wherein the time from collection of the sample to quantitation is less than about 12 hours.
14. A high throughput method for rapidly quantitating amplification products to determine the quantity of microorganisms present in multiple blood samples comprising repeated cycles of the method of claim 1 .
15. A method for rapidly quantifying multiple different microorganisms in a biological sample containing nucleic acid comprising:
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 of the biological sample with pairs of PCR primer sequences that are specific to each different microorganism and a PCR-ready composition to form a reaction mixture, wherein the PCR-ready composition comprises as components a heat-stable polymerase; a mix of deoxynucleotide tri phosphates; a chelating agent; an osmolarity agent; an albumin; at least two salts at least one of which is a magnesium salt; and a buffer present at a pH of about 6.5 to about 9.0, wherein the components are combined with nuclease-free water;
performing PCR on the reaction mixture to form amplification products that are specific for each of the different microorganisms;
detecting the quantity of each amplification product; and
determining the quantity of each microorganism in the sample, wherein the time from collection of the sample to determining the quantity of each microorganism is less than about two days.
16. The method of claim 15 , wherein each amplification product is compared with a control nucleic acid to determine the quantity of each microorganism in the sample.
17. The method of claim 15 , wherein the quantity of each amplification product provides a measure of the severity or level of infection attributable to each of said different microorganisms.
18. The method of claim 15 , wherein the biological sample is obtained from an individual and the quantity of each amplification product provides a measure of the disease state of the individual attributable to each of said different microorganisms.
19. A high throughput method for rapidly determining the quantity of each microorganism in the sample comprising the method of claim 15 .
20. A method of tracking the progression of an infectious disease of an individual wherein the infectious disease is attributable to a microorganism comprising:
obtaining multiple blood samples from the individual over a period of time;
combining at least a portion of nucleic acid of each blood sample with pairs of PCR primer sequences that are specific to the microorganism and a PCR-ready composition to form a reaction mixture, wherein the PCR-ready composition comprises a heat-stable polymerase; a mix of deoxynucleotide tri phosphates; a chelating agent; an osmolarity agent; an albumin; at least two salts at least one of which is a magnesium salt; and a buffer present at a pH of about 6.5 to about 9.0, wherein the components are combined with nuclease-free water;
performing PCR on the reaction mixture to form amplification products that are specific for the microorganism;
detecting the quantity of microorganisms present in each sample; and
determining the progression of the infection disease of the individual.
21. The method of claim 20 , wherein the individual has multiple infections attributable to multiple microorganisms and tracking the progression of each infectious disease.
22. The method of claim 21 , wherein the multiple infections include at least two of MTB, Plasmodium and HIV.
23. A high throughput method for rapidly determining the severity, progression or resolution of the infectious disease of the individual comprising the method of claim 20 .
24. A kit comprising the collection and PCR-Ready compositions of claim 1 , each contained within a sterile vessel configured for addition of a biological sample and thermal cycling, and instructions for determining the presence or absence of a pathogen from the results of the thermal cycling.
25. A method for rapidly determining the development of an infection of an individual comprising:
contacting each of multiple biological samples collected from the individual suspected of containing an infectious microorganism over multiple periods of time with a composition to form multiple reaction mixtures, wherein each reaction mixture represents one of the multiple periods of time and the composition comprises 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 at least one of which is a magnesium salt, and a buffer present at a pH of about 6.5 to about 9.0, which are combined with nuclease-free water;
performing a PCR on each reaction mixture 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 of the DNA sequences and the amplification products of the genomic sequence in each reaction mixture; and
determining a ratio of amplification products from the DNA sequences and amplification products from genomic sequences to determine the development of the infection.
26. The method of claim 25 , wherein the infection is bacterial, viral, fungal, parasitic, or a combination thereof.
27. The method of claim 26 , wherein the bacterial infection is Mycobacteria tuberculosis.
28. The method of claim 26 , wherein the viral infection is influenza virus, HIV or Zika virus.
29. The method of claim 26 , wherein the parasitic infection is Plasmodium falciparum.
30. The method of claim 25 , wherein the multiple biological samples comprise blood, serum, sputum, saliva, nasal discharge, biopsied material, or skin scraping.
31. The method of claim 25 , the multiple periods of time comprise hours, days or weeks.
32. The method of claim 25 , wherein a decrease in the quantity or ratio of amplification products of the DNA sequences as compared to the amplification products of the genomic sequences over time indicates a reduction of the development of the infection and an increase in the quantity or ratio of amplification products of the DNA sequences as compared to the amplification products of the genomic sequences over time indicates an increase of the development of the infection.
33. The method of claim 25 , further comprising administering a pharmaceutical agent to the individual.
34. The method of claim 33 , wherein the pharmaceutical agent is an antibody, a drug, an antibiotic, a natural product, a manufactured product, an antimicrobial agent, a placebo or a combination thereof.
35. The method of claim 1 , wherein the composition denatures proteins, inactivates nucleases, and kills pathogens, and does not interfere with the PCR amplifying.
36. The method of claim 15 , wherein the composition denatures proteins, inactivates nucleases, and kills pathogens, and does not interfere with the PCR.
37. The method of claim 25 , wherein the composition denatures proteins, inactivates nucleases, and kills pathogens, and does not interfere with the PCR.