A Visual Array For The Detection Of Human Papilloma Virus

Backround:Human papillomavirus (HPV) is closely associated with development of cervical cancer and its precancerous lesions. Persistent infection with high-risk HPV genotypes appears to be a prerequisite for development of cervical carcinoma. The detection of HPV infection is very important for screening of cervical carcinoma and its precancerous lesions and judgement of prognosis of the disease. For the simultaneously, sensitive and specific visual detection of 13 Human papilloma virus (HPV) types, a qualitative DNA chip method, combining multiplex asymmetrical PCR and a gold label silver stain indicator system was developed.Method:After pretreatment of infected patients samples and extract the DNA virus genome, asymmetrical PCR was performed in a single tube by using the general primer (GP5+/6+) pairs.5,-end-amino-modified oligonucleotides, which were immobilized on glass surface, acted as capturing probes that were desed toign bind single-stranded complementary targets DNA.3,-end-thiol-modified oligonucleotides, which were linked with colloidal gold,acted as detection probes that were also designed to bind complementary targets DNA. The black image of microarray spots, resulting from the precipitation of silver onto nanogold particles bound to detection probes, were used to detect targets DNA which were bound to capturing probes with a visible light scanner. Multiplex asymmetrical PCR of Human papilloma virus (HPV) and Humanβ-globin gene (used as positive control) was performed to prepare abundant single-stranded targets DNA, which affected detection efficiency and sensitivity of hybridization on chip. Plenty of clinical samples of Human papilloma virus from infected patients were tested using DNA chip.Result:1. We useβ-globin gene as a positive control, there are no marked difference between visual array and caryologia array in 100 clinical samples.2. The best concentration of capturing probe is 1μM-5μM.3. The best proportion of GP5/GP6 is 1:20. 4. The actual detection of virus is 10-9, the time of dyeing is 10 min.5. The amount of primers labeled with nanogold is 0.4μg/mL...