Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-10-25
2001-07-24
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024310
Reexamination Certificate
active
06265154
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to human papillomaviruses and, in particular, it relates to oligonucleotides for detecting human papillomaviruses in a test sample.
BACKGROUND OF THE INVENTION
To date, approximately seventy different human papillomavirus (HPV) types have been discovered. HPV is interesting from a diagnostic standpoint because several of the presently known HPV types have been linked to the development of cervical cancer. As with any form of cancer, early detection is critical to successfully treating the disease. Because certain HPV strains are associated with the development of cervical cancer, detecting HPV in an appropriate sample may provide the best means for the early detection of cervical cancer.
The polymerase chain reaction in combination with Southern blot analysis has been the prevailing method for detecting particular types of HPV in a test sample. In particular Snijders, P. J. F., et. al., J. of Gen. Virol., Vol. 71, pp.173-181 (1990) exemplifies such technology where amplification primers are employed to generate multiple copies of a sequence within the HPV genome and radiolabeled DNA probes specific for a particular HPV type are employed to detect and thereby determine the particular HPV type present in the test sample. Unfortunately, Southern blotting is a relatively labor intensive and time consuming process especially when attempting to detect multiple different HPV types. Accordingly, there is a need for methods and reagents suitable for quickly and accurately determining whether or not one or several of the HPV types associated with cervical cancer are present in a test sample.
SUMMARY OF THE INVENTION
The present invention provides oligonucleotides that can be used to specifically detect oncogenic HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 (hereinafter “oncogenic HPV types”). These oligonucleotides are designated SEQ ID NO 4 and its complement SEQ ID NO 5; SEQ ID NO 7 and its complement SEQ ID NO 8; SEQ ID NO 10 and its complement SEQ ID NO 11; SEQ ID NO 13 and its complement SEQ ID NO 14; SEQ ID NO 16 and its complement SEQ ID NO 17; SEQ ID NO 19 and its complement SEQ ID NO 20; SEQ ID NO 22 and its complement SEQ ID NO 23; SEQ ID NO 25 and its complement SEQ ID NO 26; SEQ ID NO 28 and its complement SEQ ID NO 29; SEQ ID NO 31 and its complement SEQ ID NO 32; SEQ ID NO 34 and its complement SEQ ID NO 35; SEQ ID NO 37 and its complement SEQ ID NO 38; as well as SEQ ID NO 40 and its complement SEQ ID NO 41. Preferred are cocktails of these probes comprising two or more of the above oligonucleotides.
Preferably, the oligonucleotides are employed as hybridization probes to hybridize with and detect target sequences for which they are specific. Thus, methods provided by the present invention include hybridization assays as well as amplification based assays. According to one method, a method of detecting the presence of at least one oncogenic HPV type in a test sample comprises the steps of (a) contacting the test sample with one or more of the sequences listed above; and (b) detecting hybridization between at least one of the above sequences and an oncogenic HPV target sequence as an indication of the presence of at least one oncogenic HPV type in the test sample.
According to another embodiment, a method for detecting the presence of at least one oncogenic HPV type in a test sample comprises the steps of (a) forming a reaction mixture comprising nucleic acid amplification reagents, a test sample containing an oncogenic HPV target sequence, at least one (and preferably two) primer(s) capable of amplifying an HPV target sequence designated herein as SEQ ID NO.3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 27, SEQ ID NO. 30, SEQ ID 35 NO. 33, SEQ ID NO. 36, and SEQ ID NO. 39 and one or more oligonucleotides selected from the group consisting of SEQ ID NO 4, SEQ ID NO 7, SEQ ID NO. 10, SEQ ID NO 13, SEQ ID NO 16, SEQ ID NO 19, SEQ ID NO 22, SEQ ID NO 25, SEQ ID NO 28, SEQ ID NO 31, SEQ ID NO 34, SEQ ID NO 37, SEQ ID NO 40, and their respective complements; (b) subjecting the mixture to hybridization conditions to produce at least one nucleic acid sequence complementary to the target sequence; (c) hybridizing one or more oligonucleotides to the nucleic acid sequence complementary to the target sequence, so as to form at least one complex comprising the oligonucleotide and the complementary nucleic acid sequence; and (d) detecting the so-formed complex as an indication of the presence of at least one oncogenic HPV type in the sample.
According to another embodiment, the invention provides kits which comprise a set of oligonucleotide primers, amplification reagents and at least one, and preferably at least two, of the oligonucleotides designated as SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 40 and SEQ ID NO. 41.
DETAILED DESCRIPTION OF THE INVENTION
As previously mentioned, the present invention provides oligonucleotides (hereinafter “oligos” or “probes”), methods for using the probes and kits containing the probes, all of which can be employed to specifically detect oncogenic HPV types (i.e. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). The probes provided herein can be employed as primers in an amplification reaction but preferably are employed as hybridization probes because each of the probes is specific for at least one HPV type and in one case (SEQ ID NO. 34) two HPV types. Advantageously, all of the probes hybridize within an approximately 140 bp region of the L1 gene found in the HPV genome. Thus, while the probes individually can be used to detect the oncogenic HPV type(s) for which they are specific, a cocktail comprising two or more of the oligos can be employed to detect several HPV types at once. This is particularly advantageous in an amplification reaction setting where all, more or part of the approximately 140 bp region can be amplified and the amplified product can be contacted with a cocktail of probes to determine the presence of at least one of the oncogenic HPV types in the test sample. Accordingly, a single amplification reaction can be the basis for detecting multiple HPV types. Table 1 below provides the SEQ ID NOs. of the oligos provided herein, the sequences and the HPV type(s) that they specifically detect.
SEQ ID
SEQUENCE
HPV TYPE
NO.
5′ -> 3′
SPECIFICITY
4
GCTGCCATAT CTACTTCA
16
5
TGAAGTAGAT ATGGCAGC
16
7
GTAGCATCAT ATTGCC
18
8
GGCAATATGA TGCTAC
18
10
GCAATTGCAA ACAGTGAT
31
11
ATCACTGTTT GCAATTGC
31
13
ATGCACACAA GTAACTAGT
33
14
A6TAGTTACT TGTGTGCAT
33
16
CTGCTGTGTC TTCTAGTG
35
17
CACTAGAAGA CACAGCAG
35
19
CTCTATAGAG TCTTCCATAC C
39
20
GGTATGGAAG ACTCTATAGA G
39
22
CTACACAAAA TCCTGTG
45
23
CACAGGATTT TGTGTAG
45
25
CGGTTTCCCC AACAT
51
26
ATGTTGGGGA AACCG
51
28
GTGCTGAGGT TAAAAAG
52
29
CTTTTTAACC TCAGCAC
52
31
CTACAGAACA GTTAAGTAA
56
32
TTACTTAACT GTTCTGTAG
56
34
AACTAAGGAA GGTACAT
58/33
35
ATGTACCTTC CTTAGTT
58/33
37
CTACTACTCT CTATTCCTAA TG
59
38
CATTAGGAAT AGAGAGTAGT AG
59
40
CTTTGTCTAC TACTACTGA
68
41
TCAGTAGTAG TAGACAAAG
68
The probes disclosed herein may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or nucleic acid analogs such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and 5,142,047 all of which are herein incorporated by reference. Such sequences can routinely be synthesized using a variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional
Abravaya Klara
Gorzowski Jacek J.
Hoenle Robert J.
Kroeger Paul E.
Moore Jennifer J.
Abbott Laboratories
Arthur Lisa B.
Schodin David J.
Yasger Paul D.
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