NMR systems and methods for the rapid detection of analytes
This invention features systems and methods for the detection of analytes, and their use in the treatment and diagnosis of disease.
1. A method for detecting the presence of a bacterium in a whole blood sample, the method comprising:
(a) providing an extract produced by lysing the red blood cells in a whole blood sample from a subject, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet prior to resuspending the pellet and optionally repeating the centrifuging, discarding, and resuspending steps;
(b) lysing cells in the extract to form a lysate;
(c) amplifying a bacterial target nucleic acid in the lysate to form an amplified lysate solution;
(d) following step (c), adding to the amplified lysate solution from 1×10 6 to 1×10 13 magnetic particles per milliliter of the amplified lysate solution to form a mixture, wherein the magnetic particles have a mean diameter of from 700 nm to 950 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of a target nucleic acid or a multivalent binding agent, wherein said magnetic particles have a T 2 relaxivity per particle of from 1×10 9 to 1×10 12 mM −1 s −1 ;
(e) providing the mixture in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the mixture to a bias magnetic field created using one or more magnets and an RF pulse sequence;
(f) exposing the mixture to a bias magnetic field and an RF pulse sequence;
(g) following step (f), measuring the signal from the detection tube;
(h) on the basis of the result of step (g), detecting the target nucleic acid, wherein step (g) is carried out without any prior purification of the amplified lysate solution; and
(i) on the basis of the result of step (h), determining whether the bacterium was present in the sample.
2. The method of claim 1 , wherein said whole blood sample is from 0.05 to 4.0 mL.
3. The method of claim 1 , wherein the magnetic particles are substantially monodisperse.
4. The method of claim 1 , wherein step (g) comprises measuring the T 2 relaxation response of the mixture, and wherein increasing agglomeration in the mixture produces an increase in the observed T 2 relaxation time of the mixture.
5. The method of claim 1 , wherein the amplifying of step (c) comprises amplifying a nucleic acid to be detected in the presence of a forward primer and a reverse primer, each of which is universal to multiple bacteria to form a solution comprising a bacterial amplicon; and said magnetic particles of step (d) have a first probe and a second probe conjugated to their surface, the first probe operative to bind to a first segment of the target nucleic acid and the second probe operative to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid.
6. The method of claim 1 , wherein steps (a) through (i) are completed within 3 hours.
7. The method of claim 5 , wherein the magnetic particles comprise two populations, a first population bearing the first probe on its surface, and a second population bearing the second probe on its surface.
8. The method of claim 1 , wherein said magnetic particles comprise one or more populations having a first probe and a second probe conjugated to their surface, the first probe operative to bind to a first segment of the bacterial amplicon and the second probe operative to bind to a second segment of the bacterial amplicon, wherein the magnetic particles form aggregates in the presence of the bacterial amplicon.
9. The method of claim 1 , wherein said magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and said multivalent binding agent comprises a first probe and a second probe, the first probe operative to bind to said first binding moiety and the bacterial amplicon, the second probe operative to bind to a second binding moiety and the bacterial amplicon, and the binding moieties and multivalent binding agent operative to alter an aggregation of the magnetic particles in the presence of the bacterial amplicon.
10. The method of claim 1 , wherein the amplified lysate solution of step (c) comprises whole blood proteins and non-target oligonucleotides.
11. The method of claim 1 , wherein said magnetic particles have a mean particle diameter between 700 and 900 nm.
12. The method of claim 11 , wherein said magnetic particles have a mean particle diameter of between 700 and 850 nm.
13. The method of claim 1 , wherein the target nucleic acid is derived from a bacterium selected from Acinetobacter sp., Bacteroides fragilis, Burkholderia cepacia, Campylobacter jejuni/coli, Clostridium pefringens , coagulase-negative Staphylococcus sp., Enterobacter aeraogenes, Enterobacter cloacae , Enterobacteriaceae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Haemophilus influenzae, Kingella kingae, Klebsiella oxytoca, Klebsiella pneumoniae, Listeria monocytogenes , Methicillin-resistant Staphylococcus aureus (MRSA), Morganella morganii, Neisseria meningitidis , non-meningitidis Neisseria sp., Prevotella buccae, Prevotella intermedia, Prevotella melaninogenica, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus haemolyticus, Stenotrophomonas maltophilia, Staphylococcus saprophyticus, Streptococcus agalactie, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes , and Streptococcus sanguinis.
14. The method of claim 13 , wherein said bacterium is selected from the group consisting of Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter sp., and Pseudomonas aeruginosa.
15. The method of claim 1 , wherein said lysing of step (b) comprises combining said extract with beads to form a mixture and agitating the mixture to form said lysate, which contains both subject cell nucleic acid and bacterial nucleic acid.
16. The method of claim 13 , wherein the bacterium is Escherichia coli.
17. The method of claim 1 , wherein the bacterium is Borrelia burgdorferi.
18. The method of claim 1 , wherein the method comprises individually detecting three or more of coagulase-negative Staphylococcus, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli , and Klebsiella pneumoniae.