Method for multiplexed detection of nucleic acids using spectrally encoded beads
Methods for characterizing a population of microbes in a sample are described. The methods include: amplifying microbial polynucleotides obtained from the sample to form a plurality of amplicons and combining the amplicons with a plurality of microbeads, wherein each of the microbeads has a lanthanide spectral signature paired with the sequence of capture polynucleotides immobilized on the microbead. At least some of the capture polynucleotides comprise a sequence substantially complementary to a microbe-identifying sequence in one or more amplicons, such that at least some amplicons are captured onto beads by the capture polynucleotides and the microbe can be identified based on the lanthanide spectral signature of with which the capture polynucleotide is paired. Further described are methods for the identification of pathogens present in a sample by evaluating patterns of hybridization of a capture oligonucleotide to amplicons. Microbead compositions and methods for the preparation thereof are also described.
1 . A method for characterizing a population of microbial strains in a sample, wherein different microbial strains in the population comprise polynucleotides with different microbe-identifying sequences, the method comprising:
(i) amplifying polynucleotides obtained from the sample to form a plurality of amplicons, wherein the amplicons comprise microbe-identifying sequences;
(ii) combining the amplicons with a plurality of microbeads,
wherein each microbead of the plurality of microbeads has a lanthanide spectral signature and a plurality of copies of a capture polynucleotide immobilized on the microbead,
wherein each of the plurality of copies of the capture polynucleotide immobilized on any one microbead of the plurality of microbeads comprises a same predetermined sequence
that is paired with the lanthanide spectral signature of the one microbead, such that the plurality of copies of the capture polynucleotide immobilized on the one microbead can be identified based on the lanthanide spectral signature of the one microbead, wherein the plurality of microbeads comprises one or more subpopulations, each characterized by a particular lanthanide spectral signature, such that microbeads of different subpopulations have different lanthanide spectral signatures,
wherein, for each microbead of at least some of the plurality of microbeads, the plurality of copies of the capture polynucleotide immobilized on the microbead comprises a sequence substantially complementary to a microbe-identifying sequence of one or more of the plurality of amplicons,
wherein the combining is conducted under conditions in which at least some of the plurality of amplicons hybridize to the plurality of copies of the capture polynucleotide immobilized on at least some of the plurality of microbeads, thereby producing captured amplicons, and
wherein the plurality of amplicons is labeled with one or more signal-generating moieties prior to, simultaneously with, or after being captured onto at least some of the plurality of microbeads, such that microbeads comprising immobilized signal-generating moieties are produced; wherein the magnitude of signal detected from an individual microbead corresponds to the amount of the plurality of amplicons captured on the individual microbead,
(iii) measuring the magnitude of the signal from the microbeads comprising immobilized signal-generating moieties;
(iv) for each of the plurality of microbeads, determining the lanthanide spectral signature, thereby determining the microbe-identifying sequences of the captured amplicons;
(v) generating a pattern of measured amount of hybridization of the captured amplicons to individual capture polynucleotides;
(vi) for each microbial strain of a plurality of microbial strains, comparing the pattern of hybridization measured in (v) to a predicted pattern of hybridization of amplicons to each of the individual capture polynucleotides, the predicted pattern of hybridization corresponding to the microbial strain, wherein predicting hybridization of the amplicons to a capture polynucleotide comprises calculating the Gibbs free energy of hybridization;
(vii) correlating the pattern of hybridization measured in (v) to the predicted pattern of hybridization for each microbial strain of the plurality of microbial strains to identify the microbial strain that has a predicted pattern of hybridization that has the strongest correlation with the pattern of hybridization measured in (v); and
(viii) determining that the microbial strain identified in (vii) is present in the population of microbial strains in the sample if the strongest correlation is greater than a predetermined threshold.
2 . The method of claim 1 , wherein the plurality of microbeads comprises at least 50 different spectral signatures and immobilized capture polynucleotides comprising at least 50 different predetermined sequences; and/or
the combining in step (ii) comprises hybridizing amplicons to at least 50 microbeads having different spectral signatures and different capture polynucleotides.
3 . The method of claim 1 , wherein the one or more signal-generating moieties produce a fluorescent or chemiluminescent signal; and/or the plurality of amplicons is labeled during the amplifying in step (i).
4 . The method of claim 1 , wherein at least one of the microbe-identifying sequences in the amplicons comprises a bacterial 16S ribosomal RNA (rRNA) gene sequence.
5 . The method of claim 4 , wherein the amplification in step (i) is conducted using primer pairs that hybridize to conserved regions flanking one or more variable regions in a bacterial 16S rRNA gene sequence.
6 . The method of claim 4 , wherein the amplification in step (i) comprises amplifying one or more of a V3 variable region, a V4 variable region, or a V6 variable region in a bacterial 16S rRNA gene sequence.
7 . The method of claim 1 , wherein the population of microbial strains comprises one or more species selected from the group consisting of Pseudomonas, Streptococcus, Staphylococcus, Neisseria, Acinetobacter, Escherichia, Enterobacter, Klebsiella, Haemophilus, Proteus, Serratia, Enterococcus , and Listeria.
8 . The method of claim 1 , wherein each lanthanide A spectral signature comprises a europium (Eu) signal, a dysprosium (Dy) signal, a samarium (Sm) signal, a cerium (Ce) signal, a terbium (Tb) signal, a lanthanum (La) signal, a praseodymium (Pr) signal, a neodymium (Nd) signal, a gadolinium (Gd) signal, a holmium (Ho) signal, an erbium (Er) signal, a thulium (Tm) signal, an ytterbium (Yb) signal, or a combination thereof.
9 . The method of claim 1 , wherein the one or more signal-generating moieties comprise fluorescent labels.
10 . The method of claim 1 , wherein the sample is from blood, cerebrospinal fluid, lymph, or urine.
11 . The method of claim 1 , wherein step (iii) occurs prior to step (iv).
12 . The method of claim 1 , wherein step (iii) occurs simultaneously with step (iv).
13 . The method of claim 1 , wherein step (iii) occurs after step (iv).
14 . The method of claim 1 , wherein each of the plurality of microbeads comprises a plurality of lanthanide nanoparticles.
15 . The method of claim 14 , wherein the plurality of lanthanide nanoparticles comprises a lanthanide-doped host lattice.
16 . The method of claim 15 , wherein each of the plurality of microbeads further comprises a crosslinked polymer, and wherein the capture polynucleotides are covalently bonded to the crosslinked polymer.
17 . The method of claim 1 , wherein the plurality of microbeads is dispersed on a microscope slide prior to step (iii) or step (iv).
18 . The method of claim 1 , further comprising separating uncaptured amplicons from the plurality of microbeads.
19 . The method of claim 6 , further comprising separating the microbeads comprising immobilized signal-generating moieties from microbeads not comprising immobilized signal-generating moieties.
20 . The method of claim 6 , wherein the amplification step (i) comprises amplifying the V3 variable region and the V6 variable region.