| Cervical cancer is the second largest cancer threatening women's health.Human papillomavirus(HPV)can be detected in tumor tissue in more than 90%of cervical cancer patients,indicating that detection of HPV virus DNA is an effective way to prevent cervical cancer.HPV can be divided into high risk type and low risk type according to the ability of HPV to induce cervical cancer,while persistent infection of high risk type HPV is more likely to suffer from cervical cancer.Based on the analysis of 3576 HPV-positive patients in the General Hospital of the Eastern Theater Command,18 HPV types with high risk and 5 HPV types with low risk were screened out,and the characteristics of infection distribution,age difference and infection pattern of different HPV types were analyzed.Among the12,000 subjects,the positive rate was 29.8%(3576/12000),and the main genotypes were HPV52(19.63%),HPV16(14.65%),HPV58(14.12%),HPV53(12.28%)and HPV81(11.52%).The age group with the highest HPV infection rate was?20 years old,followed by 51?60 years old,and the lowest was 31?40 years old.Among the HPV infection modes,single infection was the main type,and the rate of single infection was 62.14%.The multiple infection rate was 37.86%,and most of them were double infection.HPV infection rate(P<0.0001)and infection pattern(P<0.0001)were statistically different between age groups.Through case statistics,we found that the positive rate of multiple infections accounted for a certain proportion,and there was a statistical difference between single infection and multiple infection in different age groups.In view of the shortcomings of existing multiple infection methods,this study established a multiple detection method based on CRISPR-Cas9 technology and homogeneous nucleic acid detection(CTPCR4.0),using two or more pairs of Cas9/sg RNA complexes to cut multiple targeted DNA.The main contents of the study include:(1)The cutting segment is optimized so that the length of the segment is evenly distributed,showing multiple results.(2)Validation of two-fold to ten-fold systems and optimization of reaction systems and reaction conditions for multiple systems.(3)Validation of the feasibility and accuracy of multiple nucleic acid detection methods using 10 plasmids and clinical samples.After making different combinations,we determined the best reaction system for multiple assays:the sg RNA?insertion primers were 1?M,the universal primers was2?M,the Cas9 nuclease was 1?M.The optimum reaction conditions were as follows:the renaturation temperature was 60?and the extension time was 60 s,30 cycles.In order to meet the clinical needs,we established the blind test of the 10-fold system,and verified the specificity of the blind test of 10 kinds of HPV plasmids.Meanwhile,the sensitivity of the multiple HPV blind test system was determined to be10-3ng/ul.Finally,nucleic acid detection was carried out using the g DNA of 30 clinical samples,and 25 clinical samples were successfully detected.There were 15 cases of single infection,6 cases of double infection,2 cases of triple infection,1 case of quadruple infection and 1 case of five infection.The results of this study were compared with those of the type 23 kit,and the positive coincidence rate was 100%except for the HPV species not involved in the study.It is verified that the method has high specificity even in the complex genomic background.In this study,the epidemiological characteristics of female HPV in Nanjing area were analyzed to provide epidemiological basis for the prevention and treatment of female HPV in Nanjing area and the effectiveness monitoring of HPV preventive vaccination in the local area.At the same time,the successful establishment of multiple detection system provides a theoretical basis for the development of nucleic acid detection and genotyping kit for human papillomavirus(HPV),and provides a new idea for multiple nucleic acid detection. |
PDF Full Text Request