Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1996-11-25
2002-03-05
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S091200, C435S091520, C536S023720, C536S024320, C536S024330
Reexamination Certificate
active
06352825
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of sample analysis to determine the presence therein of Human Papilloma Virus (HPV) genotypes by amplifying HPV DNA present in the sample with a nucleic acid amplification process, e.g. the Polymerase Chain Reaction (PCR), using general primers (GPs). More in particular, the invention relates to an analysis of cervical smears which allows cervical carcinoma-related diagnosis and prognosis wherein the analysis comprises a GP-nucleic acid amplification process, e.g. GP-PCR, to determine whether the sample contains any HPV, followed by a typing of the HPV genotype present.
BACKGROUND OF THE INVENTION
HPV comprises over 70 different epitheliotropic genotypes of which over 30 are mucosotropic. Approximately one third of these mucosotropic HPV genotypes have been isolated from or associated with cervical carcinomas (De Villiers, 1989; zur Hausen, 1991).
The PCR method has been introduced as the most sensitive method for the detection of HPV DNA in clinical specimens. However, a significant heterogeneity at the nucleotide level is found between the different HPV genotypes. This has hampered the development of a simple universal PCR test for the detection of all HPV genotypes. Despite this, HPV-PCR methods have been developed which allow the detection of a broad spectrum of mainly mucosotropic HPV genotypes (Manos et al., 1989; Gregoire et al., 1989; Snijders et al., 1990).
A combination of the general primers GP5 and GP6, originally selected from the HPV L1 region on the basis of sequence information of HPV6, HPV11, HPV16, HPV18, HPV31 and HPV33 (Snijders et al., 1990; WO 91/10675), was found to amplify target DNA of at least 27 mucosotropic HPV genotypes under conditions that allow mismatch acceptance (Van den Brule et al., 1990a, 1992; de Roda Husman et al., 1994a). The strength of this GP5/6-mediated PCR method has been substantiated further by the detection of HPV DNA in 100% of cervical scrapes classified cytomorphologically as Pap IV (carcinoma in situ) and Pap V (carcinoma) in the Netherlands (Van den Brule et al., 1991; de Roda Husman et al., 1994a). This suggests that in the Dutch population all genital high risk HPVs can be detected by this assay.
Still, using GP-PCR in routine diagnostic practice, it has been found that a small number of clinical samples gives rise to ambiguous results, reflected by GP-PCR signals that are weaker than signals obtained from 50-100 Siha cells (which contain one copy of HPV16 per cell; Van den Brule et al., 1990a). This may complicate interpretation of screening results since it is presently unknown whether the weak signals represent a cross-reaction with cellular sequences or the presence of HPV genotypes which show a reduced sensitivity in the GP-PCR. It has been shown previously that some HPV types like HPV30 are detected with a decreased sensitivity in the GP-PCR (Snijders et al., 1990), and also the recently sequenced HPV types HPV39 and HPV51, showing more than three mismatches with one of the primers, have revealed a reduction in GP-PCR sensitivity (data not shown). Furthermore, some HPV types (e.g. HPV18) give rise to additional bands in the GP5/6 PCR (Snijders et al., 1990).
Recently, several groups have found that despite the presence of primer/template mismatches, a successful amplification by PCR can be ensured by the presence of perfectly matching nucleotides at the 3′-ends of the primers (Newton et al., 1989; Sommer and Tautz, 1989; Evander and Wadell, 1991).
Moreover, it also has been found that increased primer length contributes to a more efficient amplification, probably by increasing the stability of the primer/template complex (Mack and Sninsky, 1988).
Sequence analysis of the GP5/6 PCR products of different HPV genotypes has revealed the presence of HPV-specific amino acid consensus sequences directly adjacent to the 3′-ends of GP5 and GP6 (Van den Brule et al., 1992). We investigated the utility of GP5/6 primers elongated with highly conserved sequences at their 3′-ends. These elongated primers (named GP5+ and GP6+) were tested in the PCR using a model system of cloned HPV DNAs and subsequently evaluated on cervical smears which previously showed ambiguous or negative results with the original GP5/6 assay.
The results surprisingly revealed that an elongation of GP5 and GP6 with conserved sequences at their 3′-ends can overcome reduced PCR efficiencies most likely related to the number of primer/target mismatches and increase primer-template stability. Moreover, the use of elongated GP5/6 in the PCR resulted in the clarification of HPV status in cytomorphologically normal cervical scrapes which previously showed ambiguous or negative GP-PCR results.
Another desideratum in the field of HPV detection is a means to differentiate quickly between high risk and low risk HPV types. So far, individual HPV typing has been performed on the products of nucleic acid amplification by hybridization analysis using HPV type-specific oligonucleotide probes or probes consisting of cloned HPV types, or by additional type-specific PCRs. This kind of analysis entails much work, especially if one considers that the clinician usually wants to know only whether there is a high or low risk of cervical cancer. It is known by now that only a restricted group of 15 HPV types (Nos. 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56, 58, 59, 66 and 68) is associated with cervical carcinomas and carcinomas in situ (see the review of De Villiers, 1989). In a recent study it was found that 10 different HPV types (Nos. 6, 16, 18, 31, 33, 45, 51, 52, 54 and 58) were present in PAP IV scrapes tested by GP-PCR (De Roda Husman et al., 1994b). Furthermore, preliminary results from follow up studies show that only high risk HPV types show progression from cytologically normal cervix to cervical intraepithelial neoplasia (CIN) III. Classification into HPV groups with different biological behaviour instead of individual HPV typing would be less confusing and will be appreciated by the clinician. HPV detection assays using a panel of high risk HPV probes will detect most HPV-induced carcinomas and carcinomas in situ. So, for the sake of an early detection of cervical cancer, there is a need for HPV detection assays permitting a rapid differentiation between all known high risk and low risk HPV types.
We herein describe the design and performance of type-specific oligonucleotide probes which may be used either separately or in the form of cocktails for screening the GP5+/6+ mediated DNA amplification products on high and low risk HPV genotypes.
SUMMARY OF THE INVENTION
Sequence analysis of HPV GP5/6-mediated PCR products has revealed the presence of short highly conserved sequences adjacent to the 3′-ends of both primers. Since perfect matching of 3′ primer ends is critical for an efficient PCR and elongation of primers gives an additional stabilization of primer/template complexes, part of these sequences were used to elongate GP5 and GP6 at their 3′-ends. Using reconstruction experiments with different molecularly cloned HPVs, the elongated primers (named GP5+ and GP6+) showed a clearly improved detection of especially HPV genotypes having more than 3 mismatches with one or both primers. The strength of the method was further substantiated by improved HPV detection in cytomorphologically normal cervical scrapes which showed ambiguous results in the original HPV GP5/6-mediated PCR. Also a small percentage of cytological normal scrapes which were originally HPV-negative with HPV GP5/6-mediated PCR became positive after application of the elongated GP5/6 primers.
Therefore, the invention provides a general primer pair GP5/6 which has been elongated at the 3′-ends with adjacent highly conserved sequences thereby improving HPV detection in cervical smears.
Furthermore, by computer-assisted sequence analyses of the amplication product obtained by GP5/6 and GP5+/6+ PCR, which amplification product has a length of about 150 bp, we selected
Meijer Christophorus Joannes
Snijders Petrus Josephus
van den Brule Adrianus Johannes
Walboomers Jan Marcus
Cooper & Dunham LLP
Katz Robert D.
Myers Carla J.
Stichting Researchfonds Pathologie
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