Multiplex detection of vulvovaginal candidiasis, trichomoniasis and bacterial vaginosis
Methods and compositions for detection of vulvovaginal candidiasis (VVC), trichomoniasis and bacterial vaginosis (BV) are disclosed herein. In some embodiments, the presence or absence of VVC-associated Candida, Trichomonas valginalis, and a plurality of BV-related bacteria in a sample is determined using multiplex nucleic acid-based testing methods.
1. A method to detect a plurality of BV-related bacteria in a biological sample, wherein the plurality of BV-related bacteria comprises Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, and BVAB2, comprising:
contacting said biological sample with a plurality of pairs of primers, wherein the plurality of pairs of primers comprises:
at least one pair of primers capable of hybridizing to the 16S rRNA genes of Lactobacillus crispatus and Lactobacillus jensenii , wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 14 or SEQ ID NO: 15,
at least one pair of primers capable of hybridizing to the 16S rRNA gene of BVAB2, wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 4 or SEQ ID NO: 5,
at least one pair of primers capable of hybridizing to the 16S rRNA gene of Megasphaera type 1, wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 7 or SEQ ID NO: 8,
at least one pair of primers capable of hybridizing to the vly gene of Gardnerella vaginalis , wherein each primer in said at least one pair of primers comprises a sequence selected from the group consisting of SEQ ID NOS: 10-12 or a sequence differing by only one nucleotide mismatch relative to a sequence selected from the group consisting of SEQ ID NOS: 10-12, and
at least one pair of primers capable of hybridizing to the 16S rRNA gene of Atopobium vaginae , wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 1 or SEQ ID NO: 2;
generating amplicons of the 16S rRNA sequences of Atopobium vaginae , BVAB2, Megasphaera type 1, and/or Lactobacillus crispatus and Lactobacillus jensenii , and/or amplicons of the vly gene sequence of Gardnerella vaginalis from said biological sample, if said sample comprises one or more of the BV-related bacteria; and
determining the presence or amount of one or more amplified products as an indication of the presence of BV-related bacteria in said biological sample.
2. The method of claim 1 , wherein said biological sample is a clinical sample.
3. The method of claim 1 , wherein the plurality of pairs of primers comprises a first primer comprising the sequence of SEQ ID NO: 1, a second primer comprising the sequence of SEQ ID NO: 2, a third primer comprising the sequence of SEQ ID NO: 4, a fourth primer comprising the sequence of SEQ ID NO: 5, a fifth primer comprising the sequence of SEQ ID NO: 7, a sixth primer comprising the sequence of SEQ ID NO: 8, a seventh primer comprising the sequence of SEQ ID NO: 10, an eighth primer comprising the sequence of SEQ ID NO: 11, an ninth primer comprising the sequence of SEQ ID NO: 12, a tenth primer comprising the sequence of SEQ ID NO: 14, and an eleventh primer comprising the sequence of SEQ ID NO: 15.
4. The method of claim 1 , wherein
the two primers in the pair of primers capable of hybridizing to the 16S rRNA genes of Lactobacillus crispatus and Lactobacillus jensenii have the sequence of SEQ ID NOs: 1 and 2, respectively;
the two primers in the pair of primers capable of hybridizing to the 16S rRNA gene of BVAB2 have the sequence of SEQ ID NOs: 4 and 5, respectively;
the two primers in the pair of primers capable of hybridizing to the 16S rRNA gene of Megasphaera type 1 have the sequence of SEQ ID NOs: 7 and 8, respectively;
the two primers in the pair of primers capable of hybridizing to the vly gene of Gardnerella vaginalis have the sequence of:
a) SEQ ID NOs: 10 and 12, respectively, or
b) SEQ ID NOs: 11 and 12, respectively;
and the two primers in the pair of primers capable of hybridizing to the 16S rRNA gene of Atopobium vaginae have the sequence of SEQ ID NOs: 1 and 2, respectively.
5. The method of claim 1 , wherein each primer is flanked by complementary sequences comprising a fluorophore at the 5′ end, and a fluorescence quencher at the 3′ end.
6. The method of claim 1 , wherein determining the presence or amount of one or more amplified products comprises contacting the amplified products with a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence differing by only one nucleotide mismatch relative to a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16.
7. The method of claim 6 , wherein each of the plurality of oligonucleotide probes comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16.
8. The method of claim 6 , wherein at least one of the plurality of oligonucleotide probes comprises a fluorescence emitter moiety and a fluorescence quencher moiety.
9. A composition for the detection of a plurality of BV-related bacteria, wherein the plurality of BV-related bacteria comprises Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, and BVAB2, comprising:
at least one pair of primers capable of hybridizing to the 16s rRNA genes of Lactobacillus crispatus and Lactobacillus jensenii , wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 14 or SEQ ID NO: 15,
at least one pair of primers capable of hybridizing to the 16S rRNA gene of BVAB2, wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 4 or SEQ ID NO: 5,
at least one pair of primers capable of hybridizing to the 16S rRNA gene of Megasphaera type 1, wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 7 or SEQ ID NO: 8,
at least one pair of primers capable of hybridizing to the vly gene of Gardnerella vaginalis , wherein each primer in said at least one pair of primers comprises a sequence selected from the group consisting of SEQ ID NOS: 10-12 or a sequence differing by only one nucleotide mismatch relative to a sequence selected from the group consisting of SEQ ID NOS: 10-12,
at least one pair of primers capable of hybridizing to the 16S rRNA gene of Atopobium vaginae, wherein each primer in said at least one pair of primers comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 1 or SEQ ID NO: 2; and
a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16, or a sequence differing by only one nucleotide mismatch relative to a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 13, and 16, and wherein each of the plurality of probes comprises a fluorescence emitter moiety and a fluorescence quencher moiety.
10. The composition of claim 9 , wherein
the at least one pair of primers capable of hybridizing to the 16S rRNA genes of Lactobacillus crispatus and Lactobacillus jensenii comprises a primer comprising the sequence of SEQ ID NO: 1 and a primer comprising the sequence of SEQ ID NO: 2;
the at least one pair of primers capable of hybridizing to the 16S rRNA gene of BVAB2a comprises a primer comprising the sequence of SEQ ID NO: 4 and a primer comprising the sequence of SEQ ID NO: 5;
the at least one pair of primers capable of hybridizing to the 16S rRNA gene of Megasphaera type 1 comprises a primer comprising the sequence of SEQ ID NO: 7 and a primer comprising the sequence of SEQ ID NO: 8;
the at least one pair of primers capable of hybridizing to the vly gene of Gardnerella vaginalis comprises a primer comprising the sequence of SEQ ID NO: 10 and a primer comprising the sequence of SEQ ID NO: 11; and
the at least one pair of primers capable of hybridizing to the 16S rRNA gene of Atopobium vaginae comprises a primer comprising the sequence of SEQ ID NO: 12, a primer comprising the sequence of SEQ ID NO: 14, and a primer comprising the sequence of SEQ ID NO: 15.
11. The composition of claim 9 , comprising a DNA polymerase, biovalent cations, monovalent cation potassium ions, or a combination thereof.