A New Method For Detection And Typing Of HPV Based Lateral Flow Technology

Human papillomavirus(HPV)is associated with a variety of cancers,and can directly cause cervical cancer.Cervical cancer,as a preventable cancer,is a most common female-related cancer second only to breast cancer in the world.However,there are many subtypes of HPV,and the pathogenicity of different subtypes varies greatly.Meanwhile,the distribution of HPV dominant genotypes was different among regions.Therefore,accurate screening of HPV is very important for the clinical diagnosis,development,prevention,and clinical treatment,as well as the research and use of HPV vaccine based on epidemiological investigation.However,the existing HPV detection technology still has shortcomings such as long experimental cycle,complex operation,low sensitivity and poor specificity and other limitations.In our study,we analyzed positive rate and subtypes distribution of the women with cervical cells infected by HPV,the correlation between age with positive detection rate and infection pattern from the gynecological clinic department of Jin Ling Hospital,to investigate the HPV infection of cervical cells in 7057 female patients in Nan Jing and surrounding areas.And two HPV identification methods were carried out based on these results.The first method was a lateral flow system for detecting HPV based dual-label amplification which used specific primers with special markers to label the detection targets by PCR,and detected by lateral flow paper directly.The second method was a lateral flow assay system for detecting HPV based Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR).Firstly,we amplified the detection target sequence by MY09/11 universal primer in this system,then guide the specific cut of Cas12 a by cr RNA,and activated to cleavage single-strand reporter,finally reported the detection results by lateral chromatography.In the first part,the clinical data showed that the HPV positive rate of female in Nanjing and its surrounding areas was 28.07%.The dominant high-risk types were HPV52(20.91%,414/1980),HPV16(17.53%,347/1980),HPV58(16.92%,335/1980),HPV53(14.65%,290/1980),HPV56(9.29%,184/1980),according to the positive infection rate,respectively.The highest positive rate was 35.76%(418/1169)among 51-60 years old women,and the lowest was 24.11%(514/2132)among 31-40 years old women.The average rate of single infection was 59.80%(1184/1980),the average rate of double infection was 25.40%(503/1980),and the average rate of multiple infection was 14.80%(293/1980).The dominant sub-types of high-risk HPV in Nanjing were HPV52,HPV16 and HPV58,and single infection was the main infection model,followed by double infection,and age was positively correlated with the positive rate and multiple infection rate.In the second part,we verified the stability,specificity and sensitivity of the dual-label amplified HPV molecular detection system based on lateral flow chromatography technology and the lateral flow chromatography technology detection HPV system based on CRISPR by 10 high-risk HPV plasmids.Among the 30 clinical samples,HPV was detected by the dual-label amplified HPV molecular detection system based on lateral flow technology combined with agarose gel electrophoresis including 25(25/25)single infection cases and 5(5/5)double infection cases.When CRISPR-based lateral chromatography was used to detect the same 30 clinical samples,24(24/25)single infection cases and 5(5/5)single infection cases,and 1 case was failed to be detected.In summary,two HPV nucleic acid detection methods based on lateral flow assay were developed in this study based on the investigation of HPV epidemiological survey in Nan Jing.Both two methods showed they were more simple,sensitive and were shorter time-consuming compared with the traditional HPV detection methods.It indicates that HPV detection based on lateral flow tomography is a potential promising HPV screening method,which can provide fresh ideas for clinical cervical cancer screening and diagnosis.