| Objective: Human Papillomavirus(HPV)is a kind of virus apt to spreading in people and its infection rate increases quickly in rescent years.This virus not only can spread through sexual contact but also by direct or indirect ways. HPV infection is closely related with many kinds of skin hyperplasia, polypi, precancerous and cancerous lesions. HPVs are divided into a lot of subtypes. Different HPVs have different risk for diseases. Benign and malignant lesions in head and neck, digestant, respiratory and genital systems have very close relationship with HPV such as HPV6,11,16,18,31,33,35,38 and 58. At the same time certain gene point mutation of HPV can enforce their ability of causing cancer. The latest study indicated that more than 99% female with cervical cancer were infected with HPV. Cervical cancer is a common and high malignant disease among women.The occurrence of cervical cancer is very high in China and about ten thousands new cases of cervical cancer were diagnosed in women, which is a serious risk to women's health. The process of cervical cancer is closely related with HPV and gene mutation. HPV is a heavy risk to people, but not easy to be found in early stage. Therefor,establishing a practical gene detecting assay is very significant for preventing and therapy of HPV. This study aims to establish a fast, highthroughput, automated and low price technical platform on the base of TDI-FP technology to clinical screening, early diagnosis and provide more biomolecular information and excellent methods. Methods: TDI-FP technology is a compounded reaction including PCR, probe hybridization and base incorporation with high speciality and sentivity. We estabilished and optimizeed the HPV genotyping and point mutation detecting system of HPV16E7 nt 647(29th codon) and H-ras ntl698(12th codon). The objectives of genotyping are the common HPV6,11,16,18 and 58. Firstly, the LI gene of HPV6,11,16,18 and 58 were amplified by PCR with consensus GP5+/GP6+ system. And then the PCR product was added with type-specific probe to have a hybridization reaction and terminator incorporation. The special type of HPVs were detected by FP detection.The genotyping system was applied in 182 cervical samples to investigate its characteristic. The samples with HPV16 infection were detected point mutation of HPV16E7 and H-ras genes by the point mutation detecting system on the base of HPV genotyping. We primarily verified the role of gene mutation detection for helping determine super-risk people apt to cancer. Results: This study sussessfully established the HPV genotyping and malignant point mutation detecting system with good repeatability and large market. The systems can detect common HPV6,11,16,18 and 58 types, and can find the point mutation of HPV16E7 nt647 and H-ras nt 1698. The results were completely coordinate to that of sequencing. The study indicated HPV 16 and 18 are the prominent types in cervical cancer. The mutation rate of HPV16E7 and H-ras are obviously distinguished from differentlesions.Conclusion: The detecting system established in the study can be used for screening the high risk people apt to HPV infection and also can be used to predict the risk to malignant lesions,which is considered as a high sensitive, special and high throughput system.This reseach inferred that HPV16E7 point mutation may be a high risk for maligance.Attching with the H-ras mutation at 1698 would indicate the occurance of cancer.With more specimen and area and important genes detected, this detecting system will inform us more influent knowledge related to diseases of HPV. |
