1. Field of the Invention
The present invention relates to a genotyping kit for diagnosis of human papillomavirus (HPV) infection, more specifically, to a genotyping kit for detecting human papillomaviruses from clinical samples of infected patients using a DNA chip, and a process for preparing the said DNA chip and a method for diagnosis of HPV infection using the said genotyping kit.
2. Background of the Invention
Uterine cancer includes cervical cancer, endometrial cancer, uterine sarcoma and the like. For cervical cancer, approximately 450,000 new cases occur worldwide each year and approximately 6,000 in Korea. Since the occurrence of cervical cancer (including cervical intraepithelial neoplasia) occupies 22.1% of total cancer cases in Korean women, the highest incidence with the second highest death rate, the prevention, diagnosis and treatment of cervical cancer are regarded as the most important issue in women's health.
Cervical cancer progresses through a precancerous stage, cervical intraepithelial neoplasia (CIN) known to be mainly caused by human papillomavirus (HPV) infection. Especially, infection by particular types of HPV raises the possibility of developing invasive disease. Over 70 genotypes of HPV have been identified since the recognition of HPV as the main etiological factor for cervical cancer. Certain HPV genotypes were selectively found in the lesions of specific location or progression stage, which rendered the biological diversity of HPV infection realized. Among the HPV genotypes detected in the anogenital area, over 10 genotypes have been classified as the high-risk group that are associated with an elevated risk for developing cervical cancer. Based on these findings, characterization of the biological differences of HPV infection is considered to be of significant importance to the diagnosis and prevention of cervical cancer.
For the diagnosis of cervical cancer at its early stage, Pap smear test has been most commonly used which is a cytological test performed as follows: old cells removed from the outermost layer of cells from the surface of the cervix are stained and examined for histopathological characteristics of HPV infection including koilocytosis, formation of perinuclear halo in the epithelial cells. However, due to the low diagnostic efficiency (1-15%) of Pap test together with other limitations, additional methods such as colposcopy are necessary for more dependable diagnosis. Colposcopic screening can detect HPV infection up to 70% but has disadvantages including high cost of the equipment, the need for skilled interpreters, and incapability of determining HPV genotypes to distinguish between the high-risk and low-risk infection. Therefore, efforts have been made continuously to develop techniques for the detection of HPV and identification of HPV genotypes to supplement conventional screening methods for cervical cancer and its precursors including Pap test.
The methods for detection of HPV and identification of HPV genotypes can be classified into two groups, i.e., direct detection of HPV DNA and detection of amplified HPV DNA. The methods for direct detection of HPV DNA include liquid hybridization (HYBRID CAPTURE® kit by Digene Diagnostics, Silver Spring, Md., USA), Southern blot and dot blot with HPV type-specific probes, filter in situ hybridization (FISH) and the like, and the methods for the detection of amplified DNA include type-specific PCR (polymerase chain reaction) and general-primer PCR. In particular, genotype analyses of amplified HPV DNA by general primer sets are commonly performed by employing dot blot hybridization, microtiter plate hybridization, or line probe assay. Among these methods, liquid hybridization by HYBRID CAPTURE® and line probe assay following general-primer PCR have been considered most suitable for diagnostic purposes. The line probe assay can detect about 20 different HPV genotypes by immobilized oligonucleotide probes on a nitrocellulose membrane, however, it lacks reliability due to low sensitivity and difficulties in data interpretation. Commercialized HYBRID CAPTURE® kit can detect HPV DNA in clinical samples without PCR amplification and distinguish between high-risk and low-risk HPV groups. However, the fact that HYBRID CAPTURE® kit cannot identify the genotypes of infecting HPV limits accurate risk determination since the risk factor amongst the high-risk HPV is not the same, in other words, intermediate-risk types are included in the high-risk group Moreover, the use of RNA probe may pose low stability of the kit, and also possibility of contamination cannot be excluded.
Under these circumstances, there have been strong reasons for exploring and developing a simple and accurate method for detection of HPV infection and identification of the genotype of infecting HPV.