HPV is a double-stranded DNA virus whose circular genome is approximately 8 kb long. HPV inhabits the vagina, and the infection thereof is hard to treat and is not easily made to disappear. HPV infects the epithelial cells of other mammals as well as humans, and generally induces warts, and sometimes malignant tumors, at the site of infection. HPV is detected in over 90% of condyloma accuminata cases (enlarged warts having a cauliflower-like appearance around the genitals or the anus) and almost 100% of cervical cancer cases. In particular, cervical cancer accounts for 22.1% of all cancers found in women in Korea, and is the second leading cause of cancer death among women.
Thus, establishing a method of effectively detecting HPV, which causes cervical cancer, is important for the diagnosis, prophylaxis and therapy of the disease. Also, HPV needs to be effectively detected to evaluate the efficacy and toxicity of a vaccine against HPV after vaccination.
A nucleic acid-based test for diagnosing an infectious disease employs a standard method of isolating nucleic acids from individuals and clinical materials. Since target DNA or RNA is present in clinical specimens in small amounts, several major techniques used in diagnostic laboratories are based on signal amplification and target amplification. These methods aid detection, are useful in the identification of individuals without culture, and contribute to the treatment as well as diagnosis of infectious diseases. PCR, which is a nucleic acid amplification technique (NAT), is widely used because it enables the selective amplification of specific targets, present in low concentrations, to detectable levels. In addition to the qualitative detection of viruses, quantitative determination of viral load in, clinical specimens is now realized to be of great importance with respect to the diagnosis, prognosis, and therapeutic monitoring of HPV infection (Pfaller M. A, Emer. Infect. Dis. 7, 2, 2001).
The genome of all types of HPV is divided into two major regions: early and late regions. The early region of about 4.5 kb codes for genes which are associated with functions including viral DNA replication (E1), induction or suppression of the action of DNA encoding a protein inducing malignant transformation of host cells (E2), synthesis of proteins responsible for the growth of host cells and viruses (E4), stimulation of the activity of epidermal growth factor (EGF) and colony stimulator factor (CSF) receptors (E5), and malignant transformation through permanent survival of cells, activation of oncogenes and inactivation of tumor suppressor genes (E7). In particular, the oncogenic E6 and E7 proteins, which are expressed after HPV infects the epithelial cells of a host, bind to tumor suppressor proteins of host cells, p53 and pRB, respectively, thereby inhibiting the function of the tumor suppressor proteins, leading to the transformation of infected cells, resulting in the development of tumors. The late region of 2.5 kb comprises genes coding for viral major (L1) and minor (L2) capsid proteins and a non-coding region of 1 kb, which is called the long control region (LCR) that regulates the transcription and translation of the two late genes.
With recent rapid advances in molecular biological techniques, the genetic structure of HPV has been identified, revealing genomic sequences of many genotypes of HPV. HPV is classified according to the difference in DNA sequences of E6, E7 and L1 open reading frames (ORFs). When the nucleotide sequences of the ORFs differ by more than 10%, an HPV is assigned a new genotype. HPV subtypes differ by 2% to 10%, and HPV variants differ by less than 2%.
In order to specifically detect high risk HPV types 16, 18 and 31 and a low risk HPV type 11 among a large number of HPV genotypes, which are detected in tissues of cervical cancer and carcinoma in situ, respectively, the present inventors intended to detect a gene specific to each genotype of these viruses, and selected the L1 gene as such a gene.
In order to specifically detect the HPV L1 gene, the present inventors determined the sequences of L1 genes of the HPV types 11, 16, 18 and 31, which are specifically found in Koreans, and constructed primers capable of specifically binding to the L1 gene of each HPV type. The present inventors found that when PCR was performed with the primers, each HPV genotype can be specifically detected and can be precisely quantified down to very low amounts, thereby leading to the present invention.