Nmr methods and systems for the rapid detection of bacteria

Abstract

The invention features methods, panels, cartridges, and systems for detecting pathogens and for diagnosing and treating diseases, including bacteremia and sepsis.

1 .- 35 . (canceled) 
     
     
         36 . A method for detecting the presence of a species in a liquid sample, the method comprising:
 (a) amplifying in the liquid sample a first target nucleic acid and a second target nucleic acid to form a solution comprising a first amplicon and a second amplicon, wherein each target nucleic acid is characteristic of the species to be detected;   (b) adding magnetic particles to the liquid sample to form a mixture, wherein the magnetic particles comprise binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the first amplicon or the second amplicon;   (c) 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 mixture, and having an 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;   (d) exposing the mixture to a bias magnetic field and an RF pulse sequence;   (e) following step (d), measuring the signal; and   (f) on the basis of the result of step (e), determining whether the species was present in the liquid sample.   
     
     
         37 . The method of  claim 36 , where the species is a plant species, a mammalian species, or a microbial species. 
     
     
         38 . The method of  claim 37 , wherein the species is a microbial species. 
     
     
         39 . The method of  claim 36 , wherein the first target nucleic acid is amplified in the presence of a first primer pair comprising a forward primer and a reverse primer, and the second target nucleic acid is amplified in the presence of a second primer pair comprising a forward primer and a reverse primer. 
     
     
         40 . The method of  claim 36 , the magnetic particles comprise a first population of magnetic particles conjugated to a first probe and a second probe, and a second population of magnetic particles conjugated to a third probe and a fourth probe, wherein
 the first probe and third probe are operative to bind a first segment and a second segment, respectively, of the first amplicon; and   the second probe and fourth probe are operative to bind a first segment and a second segment, respectively, of the second amplicon,   wherein the magnetic particles form aggregates in the presence of the first amplicon and form aggregates in the presence of the second amplicon.   
     
     
         41 . The method of  claim 36 , wherein step (a) further comprises amplifying a third amplicon, wherein the third amplicon comprises a nucleic acid sequence that comprises the nucleic acid sequence of the first target nucleic acid and the nucleic acid sequence of the second target nucleic acid. 
     
     
         42 . The method of  claim 41 , wherein the first target nucleic acid and the second target nucleic acid are located on a chromosome or a plasmid. 
     
     
         43 . The method of  claim 41 , wherein the first target nucleic acid and the second target nucleic acid are separated by between about 10 and about 1000 base pairs. 
     
     
         44 . The method of  claim 41 , wherein the third amplicon is produced by partial run-through of strand synthesis. 
     
     
         45 . The method of  claim 38 , wherein the method is capable of detecting a concentration of 10 colony-forming units (CFU)/mL of the microbial species in the liquid sample. 
     
     
         46 . The method of  claim 45 , wherein the method is capable of detecting a concentration of 3 CFU/mL of the microbial species in the liquid sample. 
     
     
         47 . The method of  claim 46 , wherein the method is capable of detecting a concentration of 1 CFU/mL of the microbial species in the liquid sample. 
     
     
         48 . The method of  claim 36 , wherein the steps (a) through (f) of the method are completed within 3 hours. 
     
     
         49 . The method of  claim 38 , wherein the microbial species is selected from  A. baumannii, E. faecalis, E. faecium, K. pneumoniae, P. aeruginosa, E. coli , and  S. aureus.    
     
     
         50 . The method of  claim 36 , wherein the liquid sample is selected from whole blood, urine, liquid biopsy, synovial fluid, skin biopsy, cerebrospinal fluid, sputum, gastric lavage, bronchoaveolar lavage, or homogenized tissue. 
     
     
         51 . The method of  claim 50 , wherein the liquid sample is whole blood. 
     
     
         52 . The method of  claim 51 , the method further comprising, prior to step (a), providing a whole blood sample from a subject, lysing the red blood cells in the whole blood sample, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, optionally washing the pellet, and lysing the cells in the pellet to form a lysate. 
     
     
         53 . The method of  claim 36 , wherein:
 (i) step (b) comprises adding to the liquid sample from 1×10 6  to 1×10 13  magnetic particles per milliliter of the liquid sample;   (ii) the magnetic particles have a mean diameter of from 700 nm to 950 nm;   (iii) the magnetic particles have a T 2  relaxivity per particle of from 1×10 9  to 1×10 12  mM −1 s −1 ;   (iv) wherein the magnetic particles are substantially monodisperse; and/or   (v) amplifying is performed by asymmetric polymerase chain reaction (PCR).   
     
     
         54 .- 113 . (canceled) 
     
     
         114 . A method of diagnosing a bloodstream infection or sepsis in a subject, the method comprising:
 detecting, in a liquid sample obtained from the subject, the presence of a microbial species according to the method of  claim 38 ;   wherein the presence of a microbial species in the liquid sample identifies the subject as one who may have a bloodstream infection or sepsis.   
     
     
         115 .- 122 . (canceled) 
     
     
         123 . A method of treating a bloodstream infection or sepsis in a subject, the method comprising:
 detecting, in a liquid sample obtained from the subject, the presence of a microbial species according to the method of  claim 38 , wherein the presence of a microbial species in the liquid sample identifies the subject as one who may have a bloodstream infection or sepsis; and   administering a bloodstream infection or sepsis therapy to the subject identified as one who may have a bloodstream infection or sepsis.   
     
     
         124 . The method of  claim 114 , wherein the bloodstream infection is bacteremia and/or the subject is human. 
     
     
         125 . The method of  claim 123  of, wherein the bloodstream infection is bacteremia and/or the subject is a human.