Systems and methods to assess microbiomes and treatments thereof
Systems and methods to assess the health of various microbiomes and to identity species therein are disclosed. Described assessments and identifications can inform treatment decisions if a microbiome is determined to have a less than optimal balance of bacterial species within it; the presence of one or more negative species; and/or the absence of one or more positive species.
1. A kit for assessing health of a vaginal microbiome comprising:
a first set of primers comprising forward primers and reverse primers that support amplification of total bacterial DNA present in the vaginal microbiome, wherein at least one forward primer in the first set of primers comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4, and at least one reverse primer in the first set of primers comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and/or SEQ ID NO: 58; and
a second set of primers comprising at least one forward blocking primer and at least one reverse blocking primer, wherein the at least one forward blocking primer in the second set of primers partially overlaps the binding site of a forward primer in the first set of primers, wherein the at least one reverse blocking primer in the second set of primers partially overlaps the binding site of a reverse primer in the first set of primers, wherein the 3′ end of each of the at least one forward blocking primer and the at least one reverse blocking primer in the second set of primers comprises mismatches to sequences in the Lactobacillus (LB) genome, and wherein the kit is capable of detecting one or more non-LB species DNA at a titer below 1×10 6 cells/sample in an amplification reaction.
2. The kit of claim 1 , wherein the melting temperatures of the forward and reverse blocking primers are at least 68° C.
3. The kit of claim 1 , wherein the blocking primers are selected from LBblocker3, LBB3p, LBblocker4, and LBB4p.
4. The kit of claim 1 , further comprising one or more primers selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63.