Research On Methods Related To Loop-mediated Isothermal Amplification For Detection Of Human Papillomavirus Genes

Objective:（1）To establish the detection system of the human papilloma type 58 gene based on real-time fluorescent loop-mediated isothermal amplification for its rapid detection and early diagnosis.（2）To establish a rapid and visual detection method for HPV16and HPV58,which is combined the isothermal advantage of Loop-mediated isothermal amplification with the simplicity of Lateral flow dipstick assay.Methods:（1）Primer Explorer V5 was used to design primers for the conserved genes in the L1 region of human papillomavirus type 58.The nucleic acid dye SYBR GreenⅠwas added before the reaction and the concentration of d NTPs,Mg SO₄and primers in the reaction system were optimized.The optimized concentrations were determined by real-time fluorescence amplification curve.（2）The online design software Primer was used to design specific primers and probes for the conserved regions of HPV16E6gene and HPV58 L1 gene.At the same time,biotin-labeled loop primers,FAM-labeled and BHQ1-labeled probes were needed.The LAMP-LFD method detecting for HPV16/HPV58 gene is established the specificity and sensitivity of the method were tested,and the clinical samples were used to verify the validity of the method by comparing it with real-time LAMP and clinical Laboratory results（q PCR）.Results:（1）The real-time fluorescence loop mediated isothermal amplification detection system established for human papillomavirus type 58 has excellent sensitivity and specificity,and the limit of detection is 10 copies/μL.The results from q LAMP is in line with that of q PCR.（2）The LAMP-LFD reaction system was successfully constructed,the reaction temperature is 63 degrees and the reaction time is 50 minutes by optimized of LAMP-LFD.The detection of LFD only takes 5 minutes,so it takes only 55 minutes to complete the detection;The LAMP-LFD method can specifically detect HPV16 and HPV58,which was confirmed by the negative reaction of other five types of common high-risk HPV.The sensitivity of LAMP-LFD for HPV16 and HPV58 was 100 copies/μl.The results from 50 clinical samples tested by LAMP-LFD were consistent with real-time LAMP and clinical laboratory（q PCR method）.Conclusions:（1）This study established a real-time fluorescence loop mediated isothermal amplification detection system for human papillomavirus type 58 L1 gene,which is timesaving,easy to operate,highly specific and sensitive,therefore suitable for rapid detection of clinical samples of HPV.（2）The LAMP-LFD method established in our study for detection of HPV16 and HPV58 is fast and simple,and the results are easy to be observed,which is less demanding for equipments and skills than conventional LAMP or q PCR.It is extremely suitable for point-of-care testing and HPV screening in primary hospitals and resource-poor areas.