Method and kit for characterizing microorganisms

A method of identifying a species of one or more microorganisms in a biological sample, comprising:

• providing a first set of primers for amplifying a first target sequence comprising hypervariable regions V1 and V2 but not V3 of a 16S rRNA gene;
• providing a second set of primers for amplifying a second target sequence comprising hypervariable regions V4 and V5 but not V3 of the 16S rRNA gene,
• wherein the second set of primers comprises at least one forward primer and at least one reverse primer, and
• wherein the at least one forward primer comprises a sequence from the 16S rRNA gene selected from the group consisting of SEQ ID NO:
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• extracting nucleic acids from the biological sample, the biological sample comprising the one or more microorganisms;
• amplifying the first and second target sequences from the extracted nucleic acids using the first and second set of primers;
• sequencing from 5′ to 3′ V1 and V2 of the first target sequence and sequencing from 5′ to 3′ V5 and V4 of the second target sequence to obtain one or more sequence reads of the first and second target sequences, the sequencing being based on detection of hydrogen ions released during amplification of the first and second target sequences;
• defining a first target sequence subset, wherein the first target sequence subset is part or all of the first target sequence;
• defining a second target sequence subset, wherein the second target sequence subset is part or all of the second target sequence;
• performing, by a computer, a set of alignments of the first and second target sequences by comparing each of the first target sequence subset and the second target sequence subset to a set of microbial nucleic acid sequences stored in a database;
• determining, by the computer, one or more sequence portions from among the first and second target sequence subsets that have at least one alignment match to the set of microbial nucleic acid sequences stored in the database;
• identifying, by the computer, the species and/or genus of the one or more microorganisms in the biological sample based on the at least one alignment match of the first and second target sequences to the set of microbial nucleic acid sequences stored in the database;
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• correlating, by the computer, one or more antibiotic treatments to the species and/or genus of the one or more microorganisms in the biological sample, wherein the species and/or genus of the one or more microorganisms is identified by the computer.