Dual Fluorescence-based Isothermal DNA Amplification For HPV Detection

Objective:This study intends to integrate the characteristics and advantages of isothermal amplification technology and microfluidic chip technology,and to establish an HPV typing method suitable for health and medical institutions in grassroots and underdeveloped regions.That is HPV16,52 dual fluorescence isothermal multiple-self-matching-initiated amplification(IMSA)detectio-n method to construct a digital LAMP microfluidic chip that can accurately quantify HPV16type provides a new tool for further clarifying the relationship between HPV viral load and cervical lesions.Methods:1.Development of dual fluorescence-based isothermal multiple-self-matching-initiated amplification(IMSA)assay for the detection of HPV16 and HPV52.1)The E7 gene of HPV16(the E6 gene of HPV52)was used as the target sequence in which seven distinct sites were recognized by six specially designed IMSA primers.The amplification reaction was initiated at 63?and only took one hour.Dual fluorescence-based visual product detection was achieved by using a mixed dye of HNB(Hydroxy naphthol blue)and SYBR Green I which was added into the IMSA reaction system prior to amplification.2)Detection limit of the established IMSA was evaluated using the given concentrations of plasmid DNAs inserted with the target gene sequences from HPV16 and HPV52.3)Specificity of the approach was analyzed by taking different DNAs from HPV subtypes(HPV types 6,16,18,39,52,58,and 59)as non-target templates.4)In addition,the preformation of IMSA on assaying real samples was investigated by using 103 clinical specimens of cervical scraping.2.Design and manufacture of a digital LAMP microfluidic chip that can accurate-ly quantify HPV16 type.1)Double-fluorescence LAMP amplification of HPV16 in PCR tubes and analysis of its sensitivity and specificity.2)A novel microflidic chip for accurate quantification of nucleic acids was fabricated by multilayer soft lithography and made with poly-dimethylsiloxane(PDMS).The size of chip was(60 mm?40 mm?4 mm).3)Establishing a dual-fluorescent LAMP reaction system suitable for digital microfluidic chips.4)Observe the digital LAMP microfluidic chip self-priming sample injection process.5)Specificity of the approach was analyzed by taking different DNAs from HPV subtypes(HPV types 6,16,18,39,52,58,and 59)as non-target templates.6)Accurate quantification of HPV16 plasmid DNA at a concentration of 6×10~-44 pg/?L using a digital LAMP microfluidic chip.Results:1.IMSA and the dual fluorescence-based visual product detection are successfully developed for the detection of HPV16 and HPV52 with high specificity.To achieve visual detection for clarity,the optimal mixed dye is composed of 340?M HNB and1:10000?SYBR Green I.Under the excitation of a blue light,the positive reaction emits a strong yellow-green fluorescence,but a red-orange fluorescence is presented in the negative reaction,which can be easily judged by naked eyes.The limits of detection are 60 copies/reaction and 600 copies/reaction for HPV16 and HPV52,respectively.Upon detection preformation of real sample,there is no significant difference between the proposed IMSA and PCR-membrane hybridization on detection rate.2.The detection limit of the double fluorescent LAMP for HPV16 type in the PCR tube was 4×10~-44 pg/?L,and HPV16 could be specifically detected.The fabricated microfluidic chip can realize self-priming sample injection and can realize digital LAMP detection of HPV16.To achieve visual detection for clarity,the optimal mixed dye is composed of 150?M HNB and 1:10000?SYBR Green I.Under the excitation of a blue light,the positive reaction emits a strong yellow-green fluorescence,but a red-orange fluorescence is presented in the negative reaction.The digital LAMP microfluidic chip enables the accurate quantification of HPV16 type,accurate quantification of HPV16 plasmid DNA at a concentration of 163 copies/?L at a concentration of 6×10~-44 pg/?L,and specific detection of HPV16 type.Conclusion:1.The HPV 16 and 52 type dual fluorescence-based IMSA detection methods established in this study can be used for rapid screening of HPV types 16and 52 to achieve simpler and clearer interpretation of results and suitable for primary HPV gene screening.2.This study successfully constructed a microfluidic chip that can be used for digital LAMP detection.The chip satisfies the needs of digital detection and requires no external power system and microvalve.The chip can achieve high-risk HPV16 type rapid and accurate quantitative detection,and specificity good.