Systems and methods for detecting infectious diseases
Systems, methods, and devices for detecting infections in a clinical sample are provided. Small-volume clinical samples obtained at a point-of-service (POS) location and may be tested at the POS location for multiple markers for multiple diseases, including upper and lower respiratory diseases. Samples may be tested for cytokines, or for inflammation indicators. Dilution of samples, or levels of detection, may be determined by the condition or past history of a subject. Test results may be obtained within a short amount of time after sample placement in a testing device, or within a short amount of time after being obtained from the subject. A prescription for treatment of a detected disorder may be provided, and may be filled, at the POS location. A bill may be automatically generated for the testing, or for the prescription, may be automatically sent to an insurance provider, and payment may be automatically obtained.
1. A method of detecting at least one nucleic acid disease marker in a clinical sample, comprising:
a) introducing at least a portion of said clinical sample into an automatic sample processing device configured for performing immunoassays and nucleic acid amplification assays, wherein said automatic sample processing device comprises:
i) a sample handling system configured to transport said at least a portion of the clinical sample; and
ii) a detector;
b) performing an isothermal nucleic acid amplification reaction without thermal cycling for the detection of a nucleic acid disease marker present on a target nucleic acid in the at least a portion of the clinical sample, or an aliquot thereof, wherein said target nucleic acid comprises a double-stranded nucleic acid having a first strand and a second strand and said nucleic acid disease marker comprises a template region of the target nucleic acid, wherein said performing comprises contacting the at least a portion of the clinical sample, or an aliquot thereof, with a nucleic acid amplification reagent and isothermally amplifying said nucleic acid disease marker at a high temperature without thermal cycling, wherein said nucleic acid amplification reagent comprises:
a nucleic acid polymerase having strand-displacement activity, and
a first primer and a second primer, said primers having template-binding regions and having tail regions, wherein said primer template-binding regions are complementary to at least a portion of said template region of the nucleic acid disease marker, said tail regions containing 5′ terminal nucleotides of the primers, and wherein said tail region of said first primer is complementary to said tail region of said second primer, and wherein said isothermal nucleic acid amplification reaction comprises:
1) generating an extension product of the first primer by treating the target nucleic acid with said polymerase having strand displacement activity and treating the first primer under conditions such that the template-binding region of the first primer anneals to said first strand of the target nucleic acid;
2) generating an extension product of the second primer by treating the extension product of the first primer with said polymerase having strand displacement activity and treating the second primer under conditions such that the template-binding region of the second primer anneals to the extension product of the first primer;
3) repeating steps 1) and 2) to provide further extension products comprising partially double-stranded nucleic acids comprising at least one tail-region sequence of the first primer tail and comprising at least one tail-region sequence of the second primer tail, wherein said extension product comprises a double-stranded portion in which at least a portion of said first primer tail-region sequence is hybridized to at least a portion of said second primer tail-region sequence;
4) generating a concatamer strand, wherein said concatamer strand is generated as a result of the annealing of at least two hybridized tail sequences from at least two partially double-stranded nucleic acid strands to generate cross-over structures;
c) obtaining data measurements with the aid of said detector; and
d) detecting at least one nucleic acid disease marker.
2. The method of claim 1 , comprising detecting two or more nucleic acid disease markers in two or more aliquots thereof, wherein said detecting comprises isothermal amplification of two or more nucleic acid disease markers at high temperature without thermal cycling.
3. The method of claim 1 , further comprising performing an immunoassay for the detection of a further disease marker in the at least a portion of a sample, or in one or more aliquots thereof, wherein said further disease marker is other than a nucleic acid disease marker.
4. The method of claim 1 , wherein said measurements are obtained over a period of time, and wherein these measurements are indicative of the progress of the nucleic acid amplification reaction over said period of time.
5. The method of claim 1 , wherein said nucleic acid amplification is performed within a moveable assay unit.
6. The method of claim 3 , wherein said immunoassay is performed within a moveable assay unit.
7. The method of claim 3 , wherein both said nucleic acid assay and said immunoassay are performed within a moveable assay unit.
8. The method of claim 1 , wherein said sample handling system is configured to transport a moveable assay unit.
9. The method of claim 1 , wherein said clinical sample is a small-volume clinical sample having a volume of less than about 500 microliters.
10. The method of claim 1 , wherein the method is a point-of service (POS) method performed at a POS location.
11. The method of claim 10 , comprising point-of-service (POS) methods performed at a POS location, wherein the period of time from initiating said method of detecting to detecting said at least one nucleic acid disease marker is less than about 40 minutes.
12. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a nucleic acid marker selected from the group consisting of an inflammatory marker for infectious disease, an influenza marker, a marker for an upper respiratory disease, a marker for a lower respiratory disease, and a marker for a sexually transmitted disease.
13. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a nucleic acid marker for a respiratory disease selected from the group consisting of adenovirus B, adenovirus C, adenovirus E, Bordetella pertussis, mycobacterium tuberculosis (MTB), Staphylococcus aureus , Methicillin-Resistant Staphylococcus aureus (MRSA), Group A streptococcus , Group B streptococcus, Moraxella catarrhalis, Enterobacter aerogenes, Haemophilus parainfluenzae , Metapneumo Virus, Streptococcus pneumonia , Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Coronavirus OC43, Coronavirus NL63, Coronavirus MERS, Coronavirus HKU1, Coronavirus 229E, Klibsiella pneumonia phoE, Klebsiella pneumonia KPC, Bocavirus type 2,4, and Bocavirus type 1,3.
14. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a nucleic acid marker for a disease caused by a disease-causing agent selected from adenovirus B, adenovirus C, adenovirus E, Bordetella pertussis, mycobacterium tuberculosis (MTB), Staphylococcus aureus , Methicillin-Resistant Staphylococcus aureus (MRSA), Group A streptococcus , and Group B streptococcus.
15. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises an influenza marker.
16. The method of claim 15 , wherein the nucleic acid disease marker comprises an influenza marker selected from a marker for H1N1 (seasonal) influenza, H1N1 (novel) influenza, a marker for H3N2 influenza, a marker for H7N9 influenza, a marker for H5N1 influenza, and a nucleic acid sequence encoding an influenza matrix protein or portion thereof.
17. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a sexually transmitted disease.
18. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a sexually transmitted disease, wherein the disease is caused by an agent selected from the group consisting of human immunodeficiency virus (HIV), HIV-2 Group A, HIV-2, Group B, HIV-1 Group M, Hepatitis B, Hepatitis Delta, herpes simplex virus (HSV), streptococcus B, and treponema pallidum.
19. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a disease-causing agent selected from the group of disease-causing agents consisting of a virus, a bacterium, a mycoplasm, a fungus, and a yeast.
20. The method of claim 3 , comprising detecting two or more markers for disease-causing agents, wherein said markers are indicative of two or more disease-causing agents selected from a virus, a bacterium, a mycoplasm, a fungus, and a yeast.
21. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a tuberculosis ( Mycobacterium tuberculosis ) marker.
22. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a Staphylococcus bacterium.
23. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a Staphylococcus bacterium selected from a Staphylococcus aureus and Methycillin-resistant Staphylococcus aureus.
24. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a Streptococcus bacterium.
25. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a marker for a Corona virus.
26. The method of claim 3 , comprising detecting two or more markers for Corona viruses.
27. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a nucleic acid disease marker for a disease-causing agent selected from the group consisting of West Nile Virus, Epstein-Barr Virus, plasmodium, Trypanosoma cruzi , and a Dengue Virus.
28. The method of claim 3 , comprising detecting two or more markers for disease-causing agents selected from the group consisting of West Nile Virus, Epstein-Barr Virus, plasmodium, Trypanosoma cruzi , and a Dengue Virus.
29. The method of claim 1 , wherein said at least one nucleic acid disease marker comprises a nucleic acid disease marker for a disease-causing agent selected from the group consisting of Influenza A Matrix protein, Influenza H3N2, Influenza H1N1 seasonal, Influenza H1N1 novel, Influenza B, Streptococcus pyogenes (A), Mycobacterium Tuberculosis, Staphylococcus aureus (MR), Staphylococcus aureus (RS), Bordetella pertussis (whooping cough), Streptococcus agalactiae (B), Influenza H5N1, Influenza H7N9, Adenovirus B, Adenovirus C, Adenovirus E, Hepatitis b, Hepatitis c, Hepatitis delta, Treponema pallidum , HSV-1, HSV-2, HIV-1, HIV-2, Dengue 1, Dengue 2, Dengue 3, Dengue 4, Malaria, West Nile Virus, Trypanosoma cruzi (Chagas), Klebsiella pneumoniae (Enterobacteriaceae spp), Klebsiella pneumoniae carbapenemase (KPC), Epstein Barr Virus (mono), Rhinovirus, Parainfluenza virus (1), Parainfluenza virus (2), Parainfluenza virus (3), Parainfluenza virus (4a), Parainfluenza virus (4b), Respiratory syncytial virus (RSV) A, Respiratory syncytial virus (RSV) B, Coronavirus 229E, Coronavirus HKU1, Coronavirus OC43, Coronavirus NL63, Novel Coronavirus, Bocavirus, human metapneumovirus (HMPV), Streptococcus pneumoniae (penic R), Streptococcus pneumoniae (S), Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella parpertussis, Haemophilus influenzae (ampic R), Haemophilus influenzae (ampic S), Moraxella catarrhalis, Pseudomonas spp ( aeruginosa ), Haemophilus parainfluenzae, Enterobacter cloacae (Enterobacteriaceae spp), Enterobacter aerogenes (Enterobacteriaceae spp), Serratia marcescens (Enterobacteriaceae spp), Acinetobacter baumanii, Legionella spp, Escherichia coli, Candida, Chlamydia trachomatis , Human Papilloma Virus, Neisseria gonorrhoeae, plasmodium , and Trichomonas (vagin).
30. The method of claim 3 , comprising detecting two or more markers for disease-causing agents, wherein the two or more disease markers are selected from the group of markers for disease-causing agents consisting of Influenza A Matrix protein, Influenza H3N2, Influenza H1N1 seasonal, Influenza H1N1 novel, Influenza B, Streptococcus pyogenes (A), Mycobacterium Tuberculosis, Staphylococcus aureus (MR), Staphylococcus aureus (RS), Bordetella pertussis (whooping cough), Streptococcus agalactiae (B), Influenza H5N1, Influenza H7N9, Adenovirus B, Adenovirus C, Adenovirus E, Hepatitis b, Hepatitis c, Hepatitis delta, Treponema pallidum , HSV-1, HSV-2, HIV-1, HIV-2, Dengue 1, Dengue 2, Dengue 3, Dengue 4, Malaria, West Nile Virus, Trypanosoma cruzi (Chagas), Klebsiella pneumoniae (Enterobacteriaceae spp), Klebsiella pneumoniae carbapenemase (KPC), Epstein Barr Virus (mono), Rhinovirus, Parainfluenza virus (1), Parainfluenza virus (2), Parainfluenza virus (3), Parainfluenza virus (4a), Parainfluenza virus (4b), Respiratory syncytial virus (RSV) A, Respiratory syncytial virus (RSV) B, Coronavirus 229E, Coronavirus HKU1, Coronavirus OC43, Coronavirus NL63, Novel Coronavirus, Bocavirus, human metapneumovirus (HMPV), Streptococcus pneumoniae (penic R), Streptococcus pneumoniae (S), Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella parpertussis, Haemophilus influenzae (ampic R), Haemophilus influenzae (ampic S), Moraxella catarrhalis, Pseudomonas spp ( aeruginosa ), Haemophilus parainfluenzae, Enterobacter cloacae (Enterobacteriaceae spp), Enterobacter aerogenes (Enterobacteriaceae spp), Serratia marcescens (Enterobacteriaceae spp), Acinetobacter baumanii, Legionella spp, Escherichia coli, Candida, Chlamydia trachomatis , Human Papilloma Virus, Neisseria gonorrhoeae, plasmodium , and Trichomonas (vagin).
31. A method of detecting at least one nucleic acid disease marker present on a target nucleic acid, wherein said target nucleic acid comprises a double-stranded nucleic acid having a first strand and a second strand and said nucleic acid disease marker comprises a template region of the target nucleic acid, comprising:
a) introducing a cartridge into an automatic sample processing device, said cartridge comprising a clinical sample, a swab, and a nucleic acid amplification reagent, wherein:
said automatic sample processing device comprises:
i) a sample handling system configured to transport said clinical sample and for use with one or more independently movable assay units; and
ii) an optical detector; and
said nucleic acid amplification reagent comprises a nucleic acid polymerase having strand-displacement activity, a first primer and a second primer, said primers having template-binding regions, and having tail regions, wherein said primer template-binding regions are complementary to at least a portion of said template region of said nucleic acid disease marker, said tail regions containing 5′ terminal nucleotides of the primers, and wherein said tail region of said first primer is complementary to said tail region of said second primer;
b) transferring said clinical sample, or an aliquot thereof, to an assay unit for the performance of an assay for the detection of a disease marker, said transferring being performed with the aid of said sample handling system;
c) transferring said assay unit to a position suitable for detection of an optical signal from the assay unit by said optical detector;
d) performing at least one isothermal nucleic acid amplification assay at high temperature without thermal cycling for the detection of said at least one nucleic acid disease marker, wherein said isothermal nucleic acid amplification reaction comprises:
1) generating an extension product of the first primer by treating the target nucleic acid with said polymerase having strand displacement activity and treating the first primer under conditions such that the template-binding region of the first primer anneals to said first strand of the target nucleic acid;
2) generating an extension product of the second primer by treating the extension product of the first primer with said polymerase having strand displacement activity and treating the second primer under conditions such that the template-binding region of the second primer anneals to the extension product of the first primer;
3) repeating steps 1) and 2) to provide further extension products comprising partially double-stranded nucleic acids comprising at least one tail-region sequence of the first primer tail and comprising at least one tail-region sequence of the second primer tail, wherein said extension product comprises a double-stranded portion in which at least a portion of said first primer tail-region sequence is hybridized to at least a portion of said second primer tail-region sequence;
4) generating a concatamer strand, wherein said concatamer strand is generated as a result of the annealing of at least two hybridized tail sequences from at least two partially double-stranded nucleic acid strands to generate cross-over structures; and
e) detecting the at least one nucleic acid disease marker, wherein the period of time from initiating said method of detecting to detecting said at least one nucleic acid disease marker is less than about 40 minutes.
32. The method of claim 31 , comprising a first clinical sample and a second clinical sample, wherein said first clinical sample comprises a sample obtained using said swab, and said second clinical sample comprises a blood sample.
33. The method of claim 31 , comprising performing a plurality of assays for the detection of disease markers on a single small-volume clinical sample, or on aliquots thereof, and detecting two or more disease markers in said single small-volume clinical sample, or in one or more aliquots thereof.