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 kit for detecting a bacterial vaginosis (BV)-related bacteria in a biological sample, wherein the BV-related bacteria is selected from the group consisting of: Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, BVAB2, and a combination thereof, the kit comprising:
(a) 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
an oligonucleotide probe comprising a sequence of SEQ ID NO: 3, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 3,
(b) 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; and
an oligonucleotide probe comprising a sequence of SEQ ID NO: 6, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 6,
(c) 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; and
an oligonucleotide probe comprising a sequence of SEQ ID NO: 9, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 9,
(d) 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
an oligonucleotide probe comprising a sequence of SEQ ID NO: 13, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 13, and
(e) 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; and
an oligonucleotide probe comprising a sequence of SEQ ID NO: 16, or a sequence differing by only one nucleotide mismatch relative to SEQ ID NO: 16,
wherein each of the oligonucleotide probes of (a), (b), (c), (d) and (e) comprises a detectable moiety selected from the group consisting of a fluorescence emitter moiety, a fluorescence quencher moiety, a fluorophore, biotin, radionucleotides, radioactive label, and a chemiluminescent agent.
2 . The kit of claim 1 , wherein
(a) 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: 1 and a primer comprising the sequence of SEQ ID NO: 2; and
the oligonucleotide probe comprises the sequence of SEQ ID NO: 3,
(b) the at least one pair of primers capable of hybridizing to the 16S rRNA gene of BVAB2 comprises a primer comprising the sequence of SEQ ID NO: 4 and a primer comprising the sequence of SEQ ID NO: 5; and
the oligonucleotide probe comprises the sequence of SEQ ID NO: 6,
(c) 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; and
the oligonucleotide probe comprises the sequence of SEQ ID NO: 9,
(d) 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 or SEQ ID NO: 11 and a primer comprising the sequence of SEQ ID NO: 12; and
the oligonucleotide probe comprises the sequence of SEQ ID NO: 13, and/or
(e) 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: 14 and a primer comprising the sequence of SEQ ID NO: 15; and
the oligonucleotide probe comprises the sequence of SEQ ID NO: 16.
3 . A method to detect a bacterial vaginosis (BV)-related bacteria in a biological sample, wherein the BV-related bacteria is selected from the group consisting of: Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, BVAB2, and a combination thereof, comprising:
providing the kit of claim 1 ;
contacting said biological sample with the (a), (b), (c), (d) and (e) of the kit,
generating amplicons of the 16S rRNA sequences of Atopobium vaginae , BVAB2 , Megasphaera type 1, 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 one or more of the BV-related bacteria in said biological sample.
4 . The method of claim 3 , wherein said biological sample is a clinical sample.
5 . The method of claim 3 , wherein said biological sample is collected from the urethra, penis, anus, throat, cervix, or vagina.
6 . The method of claim 3 , wherein generating amplicons is carried out using a method selected from the group consisting of: polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, Immuno-amplification, nucleic acid sequence based amplification, self-sustained sequence replication, rolling circle amplification, and transcription-mediated amplification (TMA).
7 . The method of claim 6 , wherein the PCR is real-time PCR.
8 . The kit of claim 1 , further comprising a DNA polymerase and/or a plurality of dNTPs.
9 . The kit of claim 1 , wherein 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: 1 and a primer comprising the sequence of SEQ ID NO: 2.
10 . The kit of claim 1 , wherein the at least one pair of primers capable of hybridizing to the 16S rRNA gene of BVAB2 comprises a primer comprising the sequence of SEQ ID NO: 4 and a primer comprising the sequence of SEQ ID NO: 5.
11 . The kit of claim 1 , wherein 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.
12 . The kit of claim 1 , wherein 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 or SEQ ID NO: 11 and a primer comprising the sequence of SEQ ID NO: 12.
13 . The kit of claim 1 , 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: 14 and a primer comprising the sequence of SEQ ID NO: 15.
14 . The kit of claim 1 , wherein each primer is attached to a fluorophore at the 5′ end, or a fluorescence quencher at the 3′ end, or both.