Methods and compositions for rapid and high throughput diagnosis
Provided herein are methods and compositions for diagnosing a disease or an infection in a high throughput manner. Also provided herein are methods and compositions for diagnosing a disease or an infection with high specificity and high sensitivity.
1. A method of detecting a target nucleic acid sequence in a sample, said method comprising:
contacting said sample with at least one microdroplet comprising a pair of primers and a probe, wherein said at least one microdroplet comprising said pair of primers and said probe contains a volume equal to or less than 250 nL, wherein said at least one microdroplet comprises said pair of primers and said probe at a concentration of at least 1 μM; and amplifying said target nucleic acid sequence in said sample, thereby detecting said target nucleic acid sequence in said sample, wherein said amplification comprises polymerase chain reaction thermocycling.
2. The method of claim 1 , comprising contacting said sample with two or more microdroplets each comprising said pair of primers, wherein each of said two or more microdroplets has a volume of 2.5 nL or 25 nL.
3. The method of claim 1 , wherein said target nucleic acid sequence is DNA, optionally wherein said target nucleic acid sequence is viral DNA, optionally further wherein said viral DNA is from hepatitis B, adenovirus, papillomavirus, poxvirus, herpesvirus, herpes simplex virus, varicella zoster virus, Epstein-Barr virus, or cytomegalovirus.
4. The method of claim 3 , wherein said DNA comprises DNA from a bacterium, a fungus, or a parasite, optionally wherein said bacterium comprises Streptococcus pyogenes , coliform, Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Clostridium difficile, Mycobacterium tuberculosis, Bordetella pertussis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Legionella pneumophila, Neisseria meningitidis, Listeria monocytogenes, Borrelia burgdorferi, Vibrio cholerae, Clostridium botulinum, Clostridium tetani , or Bacillus anthracis , optionally wherein said fungus comprises Candida albicans, Trichophyton, Microsporum, Epidermophyton, Trichophyton rubrum, Epidermophyton floccosum, Aspergillus, Histoplasma capsulatum, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides or Blastomyces , optionally further wherein said parasite comprises a protozoa, a helminth, or an ectoparasite.
5. The method of claim 1 , wherein said target nucleic acid sequence comprises a nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), influenza virus, Dengue virus, hepatitis C virus, hepatitis E virus, ebolavirus, lyssavirus, poliovirus, West Nile virus, Human T-cell lymphotropic virus type 1 (HTLV-1), respiratory syncytial virus (RSV), parainfluenza virus (NV), human metapneumovirus (hMPV), human rhinovirus (HRV), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), middle east respiratory syndrome coronavirus (MERS-CoV), or measles virus.
6. The method of claim 1 , wherein said amplification is performed in a reaction mixture.
7. The method of claim 6 , wherein said reaction mixture has a volume of about 0.5 to about 20 μL.
8. The method of claim 6 , wherein said reaction mixture comprises a polymerase, deoxynucleotide triphosphates (dNTPs), DNAse/RNAse-free water, or an amplification buffer, optionally wherein said polymerase comprises a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
9. The method of claim 6 , wherein said reaction mixture comprises a reverse transcriptase.
10. The method of claim 1 , wherein said amplification has at least 91% sensitivity relative to a positive control amplification or at least 91% sensitivity relative to a negative control amplification.
11. The method of claim 1 , wherein said amplification has a lower limit of detection (LoD) of 10-200 molecules per mL as determined by amplification with standard positive controls.
12. The method of claim 1 , wherein said detection has a lower limit of detection (LoD) of 100-2000 molecules per mL as determined by amplification with standard positive controls.
13. The method of claim 1 , wherein said sample is extracted from a biological sample.
14. The method of claim 13 , wherein said biological sample comprises nasopharyngeal fluid, oropharyngeal fluid, saliva, blood, sera, plasma, lavage, urine, ear exudate, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, auroral pharyngeal lavage fluid, bronchoalveolar lavage, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretion, mucosal secretion, stool, stool water, pancreatic juice, lavage fluid from sinus cavities, bronchopulmonary aspirate, blastocoel cavity fluid, or umbilical cord blood, further wherein said biological sample is obtained from a human subject, optionally further wherein said biological sample is treated with N-acetylcysteine (NAC) before each of said plurality of samples is extracted from said biological sample, optionally further wherein said biological sample is heat-inactivated before each of said plurality of samples is extracted.