| Objectives Condyloma acuminatum is mainly caused by persistent infection of low-risk human papillomavirus,more than 90% of Condyloma acuminatum are associated with HPV6 and HPV11,so it is necessary to establish a simple,rapid and accurate detection method of HPV6 and HPV11.In this study,HPV6 detection method,HPV11 detection method,and HPV6/11 detection method were established by recombinant enzymemediated isothermal amplification with endogenous parameters.The sample releasing agent was used to simplify the procedure of nucleic acid treatment and shorten the extraction time.Methods 1 According to the design principle of RAA,several specific primers and probes were designed in the conservative sequence region of HPV6 and HPV11,respectively.The best primer and probe combination was selected by using recombinant plasmids,then its sensitivity and repeatability were tested.Its specificity was tested by using nucleic acids from other HPV-positive clinical samples.233 clinical samples were detected and evaluated,and Shengxiang Fluorescent PCR kits were used for parallel detection.2 Human β-globin was selected as the internal reference detection gene,and the human β-globin primer probe was combined with RAA HPV6 primer probe and RAA HPV11 primer probe to form EIC-RAA detection method.The EIC-RAA system was optimized by dynamically adjusting the amount of human β-globin primers and probes,and its specificity was tested by using nucleic acids from other HPV positive clinical samples.233 clinical samples were detected and evaluated,and Shengxiang Fluorescent PCR kits were used for parallel detection.3 Using Vector NTI software,the complete sequence of HPV6 and HPV11 was compared to find a relatively conservative region,where the universal HPV6/11 primers and probes were designed.The EIC-RAA HPV6/11 system was established by combining human β-globin primer and probe,its specificity was tested by using nucleic acids from other HPV positive clinical samples,233 clinical samples were detected and evaluated,and Shengxiang Fluorescent PCR kits were used for parallel detection.4 The sample releasing agent was used to simplify the procedure of nucleic acid extraction before EIC-RAA detection,and the sample releasing agent combined with EIC-RAA detection method was established by optimizing the use conditions of the sample releasing agent.The nucleic acid was extracted from the 233 clinical samples by this method and the magnetic beads method in parallel.The nucleic acids extracted by the two methods were detected by EIC-RAA.Results 1 HPV6-PF1R1 and HPV11-PF1R1 are the best primers probes combination for RAA detection of HPV6 and HPV11,respectively.The detection sensitivity of RAA HPV6 assay and RAA HPV11 assay was 10copies/μL,with good repeatability and specificity.Compared with Shengxiang Fluorescent PCR kits,the coincidence rate of RAA HPV6 assay and RAA HPV11 assay was 100%,and the Kappa value was 1(P < 0.001).2In EIC-RAA HPV6 and EIC-RAA HPV11,the best addition of human β-globin primer and probe was 0.3μL and 1.0 μL,and the sensitivity of EIC-RIAA HPV6 was 10copies/μL,the sensitivity of EIC-RAA HPV11 detection method was 100copies/μL,with good repeatability and specificity.Compared with Shengxiang Fluorescent PCR kits,the coincidence rate of EIC-RAA HPV6 and EIC-RAA HPV11 was 100% and the Kappa value was 1(P < 0.001).3 The RAA primers and probes of universal HPV6/11 were designed in the conservative L1 segment by sequence comparison,and the EIC-RAA detection method was established.Its sensitivity was 100 copies/μL,with good repeatability and high specificity.Compared with Shengxiang Fluorescent PCR kits,the coincidence rate of EIC-RAA HPV6/11 was 100% and the Kappa value was 1(P < 0.001).4 The best conditions for the sample release agent are 200μL concentrated solution and200μL original sample addition,and 2μL template addition.Compared with the magnetic bead method,the coincidence rate of the sample release agent combined with the EICRAA method is 100%,and the Kappa value is 1(P < 0.001).Conclusion In this study,the EIC-RAA HPV6 assay,EIC-RAA HPV11 assay,and EICRAA HPV6/11 assay have been successfully established,which have the advantages of high sensitivity,good specificity,and simple operation.Combined with the sample releasing agent,the method has the advantages of rapid and field application,and it can be used to detect HPV6 and HPV11 infection in the field under the condition of limited medical resources,rapid screening in grass-roots community hospitals.Figure 9;Table 7;Reference 103... |
PDF Full Text Request