Rapid epidemiologic typing of bacteria
View Patent ↗Methods for typing a strain of an organism are provided, the methods comprising the steps of amplifying, in a single reaction mixture containing nucleic acid from the organism, dividing the reaction mixture into a plurality of sets of second-stage reaction wells, each set of second-stage reaction wells containing a different pair of second-stage primers, subjecting each of the second-stage reaction wells to amplification conditions to generate a plurality of second-stage amplicons, melting the second-stage amplicons to generate a melting curve for each second-stage amplicon, and identifying the strain of the organism from the melting curves.
1. A method for identifying a bacterium in a sample, comprising the steps of:
obtaining the sample comprising the bacterium;
amplifying a nucleic acid from the sample, wherein the amplifying step is performed in a single reaction mixture comprising the nucleic acid from the sample and a plurality of different pairs of first-stage primers for amplifying nucleic acids from various bacterial species, each of the different pairs of first-stage primers capable of specifically hybridizing to a conserved genetic sequence of a specific gene of a particular bacterial species and amplifying a nucleic acid from the particular bacterial species, thereby generating a plurality of first-stage amplicons;
dividing the plurality of first-stage amplicons into a plurality of sets of different second-stage reaction wells, each set of the different second-stage reaction wells containing a pair of different pairs of second-stage primers, each pair of the different pairs of second-stage primers specifically hybridizing to a variable region within a specific gene of one of the first-stage amplicons of a particular bacterial species of the various bacterial species, wherein the second-stage primers are nested primers of the first-stage primers, and the different second-stage reaction wells comprise standard wells and at least one sample-only well, each of the standard wells contains a known sequenced allele within the specific gene of the particular bacterial species of the various bacterial species and the at least one sample-only well does not contain the known sequenced allele within the specific gene of the particular bacterial species of the various bacterial species;
subjecting each of the different second-stage reaction wells to an amplification reaction, thereby generating a plurality of second-stage amplicons, wherein, during the amplification reaction, in each of the standard wells, both the known sequenced alleles within the specific gene of the particular bacterial species of the various bacterial species and the first-stage amplicons are amplified, while, in the at least one sample-only well, only the first-stage amplicons are amplified; and
melting the second-stage amplicons in each of the different second-stage reaction wells after the amplification reaction, thereby generating a melting curve for each of the different second-stage reaction wells, and identifying the bacterium in the sample by comparing the melting curve of the sample-only well with the melting curve of each of the standard wells, wherein the bacterium in the sample is the particular bacterial species in one of the standard wells when the melting curve of the one of the standard wells matches the melting curve of the sample-only well and there is no detectable difference between the melting curve of the sample-only well and the melting curve of each of the standard wells.
2. The method of claim 1 , wherein the first-stage primers are degenerate primers.
3. The method of claim 1 , wherein the bacterium is S. aureus.
4. The method of claim 3 , wherein the different pairs of second-stage primers in seven of the sets of different second-stage reaction wells are used for amplifying genes arcc, aroe, glpf gmk, pta, tpi, and yquil.
5. The method of claim 1 , wherein the identifying step further includes typing a strain of the bacterium.
6. The method of claim 1 , wherein the amplifying, dividing, subjecting, melting, and identifying steps are all performed in a sealed container.
7. The method of claim 6 , wherein the identifying step is performed without sequencing or using sequence-specific probes.
8. The method of claim 1 , wherein the sample is selected from the group consisting of sputum, a nasal swab, a nasal aspirate, a tracheal aspirate, a respiratory specimen, a wound culture and a sterile site culture.
9. The method of claim 1 , wherein the melting curve of each of the standard wells is normalized before the comparing step.
10. The method of claim 1 , wherein the melting curves are plotted to visualize melting curve differences.