Establishment Of Assimilating Probes-based Real-time Duplex Loop-mediated Isothermal Amplification For Simultaneously Detection Of HPV16 And HPV 18

Objective:This study intends to construct a new amplification method based on assimilating probe and heat labile UDG enzyme of loop-mediated isothermal amplification?LAMP?for the detection of HPV16 and 18 with high sensitivity and specificity.Methods:1.Primers for the conservative sequences of HPV16 and HPV18 genes were designed by the online LAMP primer design software Primer Explorer V5.The nucleic acid dye SYBR Green I was added before the reaction.Inner primers,Mg²⁺and d NTPs in the LAMP reaction were optimized,and the optimized concentrations were determined by real-time LAMP amplification curves.Serial dilutions of plasmids of HPV16 and HPV18 were used to test the real-time LAMP sensitivities.The specificities of reactions for HPV16 and HPV18 were verified by different clinical samples subtypes.2.The oligonucleotide probe designed to specifically identify the region in the loop primer region designed for LAMP reactions?i.e.,between F1 and F2 or between B1 and B2?to generate fluorescence signals by the strand displacement reaction.The limits of detection were tested using serial dilutions of plasmids HPV16 and HPV18.And the assimilating probe-based real-time duplex LAMP was then performed using clinical samples to evaluate the specificity and the consistency with PCR results.3.A set of specific LAMP amplification primers was designed based on HPV18 L1 gene,and a UDG-LAMP reaction was preliminarily established to eliminate contamination.Results:1.The specificities of the established real-time LAMP for the detection of HPV16 and HPV18 were 100%and the coincidence rate was 100%compared with PCR.The detection limits were 10 copies/reaction both for HPV16 and HPV18.2.The assimilating probe-based duplex LAMP reaction was completed at 63?for 60 min,the amplification results could be identified based on the different fluorescence color of the two specifically amplified products.The detection limits for HPV16 and HPV18 analyzed in single LAMP reaction were less than 100 copies/reaction,and that of duplex LAMP reaction was 10 copy/reaction.In addition,as for clinical samples,the coincidence rate between the probe-based duplex LAMP reaction and PCR were95%,100%,100%for HPV16-positive,HPV18-positive and double-positive samples.3.Compared with the traditional real-time LAMP,the real-time UDG-LAMP reaction can effectively control the carryover contamination.To some extent,the false positive rate of real-time LAMP detection is reduced,and the accuracy is improved.Conclusion:1.The established real-time LAMP reactions for detecting HPV16 and HPV18 can be used for early screening of HPV infection because of simpleness,rapidness and high sensitivities.2.The assimilating probe-based duplex LAMP assay was simple,specific,and rapid,making it a useful tool for POCT setting.3.The method of eliminating contamination by heat labile UDG enzyme is helpful to promote the application in the field of real-time detection and in vitro diagnosis.