Assay for detecting closely-related serotypes of human papillomavirus (HPV)

A multiplex assay for detecting the presence or absence of at least one serotype of human papillomavirus (HPV) in a biological sample wherein signals from more than one serotype are on a single channel, comprising:

• providing a biological sample;
• contacting the biological sample with at least three oligonucleotide primer/probe sets, each of which comprises at least one oligonucleotide probe that is detectably labeled and has a nucleotide sequence length of about 10 to about 50 and at least two oligonucleotide primers each of which has a nucleotide sequence length of about 10 to about 150 under conditions such that the probes and primers anneal to a target sequence and wherein each primer/probe set is at least preferential for a specific serotype of an organism wherein the first and second primer/probe sets are degenerate with respect to each other and wherein the degenerate probes each are modified by a signal moiety that emits signal that is detectable on the same channel and wherein the third primer/probe set is not degenerate with respect to the other two primer/probe sets and discriminates for a third serotype that is not the serotypes to which the degenerate primer/probe sets preferentially anneal and wherein the probe in the third primer/probe set is modified by a signal moiety that emits signal at a wavelength that is the same or different from the wavelength emitted by the signal moiety of the degenerate probes wherein the target for the degenerate primer/probe set is SEQ ID NO:
• 37;
• further comprising contacting the biological sample with at least one additional degenerate primer/probe set wherein the target for the additional degenerate primer/probe set is SEQ ID NO:
• 36 and wherein the third primer/probe set is configured to discriminate for HPV serotype 51 and is not degenerate with respect to the first or second primer/probe set, wherein the third primer/probe set comprises at least one oligonucleotide having a sequence selected from the group consisting of SEQ ID NOS:
• 24-29 and sequences that are at least 70% homologous to at least one of SEQ ID NOS:
• 24-29;
• the first primer/probe set selects for HPV serotype 39 and does not discriminate for HPV 39 serotype with respect to HPV serotype 68, wherein the first primer/probe set comprises at least one oligonucleotide having a sequence selected from the group consisting of SEQ ID NOS:
• 1, 3 and 5 and sequences that are at least 70% homologous to SEQ ID NOS:
• 1, 3 and 5 and the second primer/probe set selects for HPV serotype 68 and does not discriminate for HPV 68 serotype with respect to HPV serotype 39, wherein the second primer/probe set comprises at least one oligonucleotide having a sequence selected from the group consisting of SEQ ID NOS:
• 2, 4, 6 and sequences that are at least 70% homologous to SEQ ID NOS:
• 2, 4 and 6;
• amplifying, if present, the target sequence;
• and
• monitoring for detection the label as an indication of the hybridization of the probe set to the target sequence thereby indicating the presence or absence of at least one of the serotypes of HPV.

A multiplex assay for detecting the presence or absence of at least one serotype of human papillomavirus (HPV) in a biological sample wherein signals from more than one serotype are on a single channel, comprising:

• providing a biological sample;
• contacting the biological sample with at least three oligonucleotide primer/probe sets, each of which comprises at least one oligonucleotide probe that is detectably labeled and has a nucleotide sequence length of about 10 to about 50 and at least two oligonucleotide primers each of which has a nucleotide sequence length of about 10 to about 150 under conditions such that the probes and primers anneal to a target sequence and wherein each primer/probe set is at least preferential for a specific serotype of an organism wherein the first and second primer/probe sets are degenerate with respect to each other and wherein the degenerate probes each are modified by a signal moiety that emits signal that is detectable on the same channel and wherein the third primer/probe set is not degenerate with respect to the other two primer/probe sets and discriminates for a third serotype that is not the serotypes to which the degenerate primer/probe sets preferentially anneal and wherein the probe in the third primer/probe set is modified by a signal moiety that emits signal at a wavelength that is the same or different from the wavelength emitted by the signal moiety of the degenerate probes wherein the target for the degenerate primer/probe set is SEQ ID NO:
• 37;
• further comprising contacting the biological sample with at least one additional degenerate primer/probe set wherein the target for the additional degenerate primer/probe set is SEQ ID NO:
• 38 wherein the third primer/probe set is configured to discriminate for HPV serotype 59 and is not degenerate with respect to the first or second primer/probe set, wherein the third primer/probe set comprises at least one oligonucleotide having a sequence selected from the group consisting of SEQ ID NOS:
• 30-35 and sequences that are at least 70% homologous to at least one of SEQ ID NOS:
• 30-35;
• the first primer/probe set selects for HPV serotype 56 and does not discriminate for HPV 66 serotype with respect to HPV serotype 56, wherein the first primer/probe set comprises at least one oligonucleotide having a sequence selected from the group consisting of SEQ ID NOS:
• 17, 19, 21 and sequences that are at least 70% homologous to SEQ ID NOS:
• 17, 19,and 21 and the second primer/probe set selects for HPV serotype 66 and does not discriminate for HPV 66 serotype with respect to HPV serotype 56, wherein the second primer/probe set comprises at least one oligonucleotide having a sequence selected from the group consisting of SEQ ID NOS:
• 18, 20, 22, 23 and sequences that are at least 70% homologous to SEQ ID NOS:
• 18, 20, 22, and 23.