Numerous papillomavirus sequences were determined, see the publications incorporated herein by reference: HPV-6: de Villiers et al., J. Virology, 40 (1981); HPV-11: Dartmann et al., Virology 151, 124-130 (1986); HPV-16: Seedorf et al., Virology 145, 181-185 (1985); HPV-18: Cole and Danos, Journal of Molecular Biology 93, 599-608 (1987); HPV-31: Goldsborough et al., Virology 171, 306-311 (1989); HPV-33: Cole and Streeck, J. Virology, 58, 991-995 (1986); HPV-54: Favre et al., J. Cancer 45, 40-46 (1990); HPV-56: Lõrincz, J., Gen. Virol. 70, 3099 (1989).
Detecting and typing of HPV is reported in a number of publications, besides Soutern blotting and other hybridisation techniques, the most widely used techniques are the PCR-based methods, since these methods simultaneously provide high sensitivity, specificity and the flexibility of the assay gives more control to comply the analytical requirements.
Human papillomavirus, a member of the Papillomaviridae family, is a DNA tumorvirus, with an 8000 bp of circular genome. The virus shows strong epithelial tropism, and proliferates only in differentiated epithelial cells. The papillomavirus has suspected etiologic role in many different human diseases, for example in different skin diseases, i.e. in verruca, condyloma acuminatum and skin tumours and in other conditions, such as cervical carcinoma, anogenital carcinomas, laryngeal carcinoma. It is well established that the human papillomavirus shows strong correlation with the incidence of these tumors, and this is even true for the pre-cancerous lesions (ClN, VlN, VAIN, PIN, PAIN). HPV can be detected in 99% of the cervical carcinoma patients. This close statistical relationship is possibly caused by the causal role of the HPV in the formation of cervical carcinoma. On the basis of the epidemiological data, the patients to be infected by different HPV genotypes do not have the same level of risk to develop cervical carcinoma. According to these findings the genotypes are classified into low risk, medium risk and high risk classes, and besides these there are not-classified genotypes too. Since the risks are grossly different and the incidence of the HPV infection is very high, the determination of genotype is of great importance.
The HPV virus can not be cultivated. The serological diagnosis of HPV infection is limited to detect the exposure to the virus (past or present infection), but can not exactly identify the genotype, the role is mostly limited to epidemiological investigations.
For papillomaviruses, exact serologic classification (serotyping) does not exist genotyping is the widely accepted classification method. These can be divided into two groups, according to whether detection is preceded by amplification or not. In one embodiment of the latter method, full length genomic RNA probes are used to detect the denatured HPV DNA genomes, and the heteroduplex is detected with specific antibodies (Hybrid Capture—Digene). According to another method, Southern blot technique is used for detection and genotyping the HPV genotypes. The disadvantage of these methods is the relative insensitivity and partial lack of specificity. In the case of the Hybrid Capture method many publications report different cross-reactions, causing false positive reactions in clinical conditions. The authors reported that the cross-reactions were acceptable only with a cut-off control of high (1 ng/ml) DNA concentration, which underlines the non-desirable coupling between sensitivity and specificity.
By the amplification methods this problem does not appear, since the reaction responsible for the sensitivity (amplification) is carried out separately.
Generally the amplification techniques differ in the selected amplified genome segment, number of primers, and the applied detection technique. The most frequently used primers are the GP5+-GP6+, MY9-MY11 and the different type-specific PCR reactions.
The most frequently used detection techniques are the sequence-specific hybridisation, restriction fragment length polymorphism (RFLP) and the line probe assay (LiPA). Besides these ones, sequencing of amplicons and thymidine pattern generated by dUTP incorporation is used, but less frequently.
The analytical characteristics of the amplification techniques vary in wide ranges. The methods can be characterized by the amplifiable genotypes, the analytical sensitivity of the genotype amplification and the specificity and reliability of the detection. In this field the MY9-MY11 degenerated primer system is considered to be the reference reaction. In case of the MY9-MY11 system LiPA hybridisation detection system exists (Innogenetics). The major drawback of the MY9-MY11 system it is difficult to control the degenerated synthesis of the primers that is why the relative ratio of the primer species produced in the synthesis is varying from synthesis to synthesis, which can result in the unpredictable changes of the analytical behaviour of the PCR reaction; secondly, this reaction can amplify the fewest types, compared to the other widespread used reactions. It is well known from the literature, that the system can amplify genotype 51 only in that case, if the HPV genotype 51 type-specific primers are added to the reaction. Using degenerate primer synthesis the relative ratio of the primer species can not be changed, and it is impossible to tailor the primer ratios to achieve better analytical performance and a balanced amplification of genotypes.
The GP5+-GP6+ reaction solves the problem only by the use of two carefully selected pair of primers—optimised to the genital HPV sequences—the two primer systems are easy to manage, however the flexibility is lower. The GP5+-GP6+ system can amplify a lot of known HPV genotypes, but the analytical characteristics of the system are not optimal (sensitivity is not balanced with different genotypes), and the two primer approach is constrained in optimisation, e.g. balancing the detection sensitivities for the individual genotypes is highly problematic (except the limited optimisation of the melting temperature and concetration of the MgCl2). It is difficult to adapt the GP5+-GP6+ system to the amplification of other genotypes, which in any case influence its future application, since the need for detecting new genotypes permanently occur. The identification of the genotypes is not solved adequately.
Another well known wide genotype-specific amplification method is the L1C method: two-primer system, with two versions, one is using (with the LC1 primer) the L1C2 or the new L1C2 primer, to amplify further genotypes. The detailed description of the L1C amplicon can be found in the literature [Jpn. J. Cancer Research 82, 524-531 (1991)].
Basically two criteria must be fulfilled by the detection postamplification methods: routine diagnostic applicability (simplicity, costs, time), and the requirement of power of discrimination suitable level of discrimination power. A significant group of methods are not suitable in terms of power of discrimination discrimination power. Therefore the application of the RFLP is limited, because of the short amplified regions, there are not enough diagnostic restriction sites, so often the genotypes can only be classified into groups. Another example the SSCP technique is difficult to refer the complex patterns of the SSCP to genotypes, and also, the robustness of these reactions is not satisfactory, either.
The power of discrimination is especially important from the diagnostic point of view to fulfil the requirements of the regulatory authorities. From the aspects of the simplicity and the power of discrimination sequencing is the ideal approach, since its automation is solved and able to detect each genotypes (or even subtypes thereof), if the sample is not a mixture of genotypes. But in the practice it is not widely used, because it is expensive and time-consuming, and its application in routine diagnostic laboratories is not acceptable, and in case of mixed samples none of the genotypes can be determined.
The advantage of the hybridisation methods is that their power of discrimination or stringency can easilybe changed, since several parameters of the reaction can be varied in wide ranges, and some forms are easily automated, the reaction is less expensive, and in case of parallel implementation (with some forms) even the time needed is insignificant.
Therefore there is a need for a new HPV amplification/detection method, which eliminates the disadvantages of the current methods, and it is cheap, easy to reproduce and automate. The invention describes an amplification and hybridisation assay, in which the primers are independently synthesized molecules, therefore their relative ratio can easily be controlled and optimised, and the amplification has a balanced sensitivity. Hybridisation reactions carried out in highly parallel manner comply with the criteria of a low cost, fast, flexible and automatable reaction.